[go: up one dir, main page]

EP3132034B1 - Therapeutic - Google Patents

Therapeutic Download PDF

Info

Publication number
EP3132034B1
EP3132034B1 EP15718982.0A EP15718982A EP3132034B1 EP 3132034 B1 EP3132034 B1 EP 3132034B1 EP 15718982 A EP15718982 A EP 15718982A EP 3132034 B1 EP3132034 B1 EP 3132034B1
Authority
EP
European Patent Office
Prior art keywords
sequence
dna
spacer
gene
antibiotic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Revoked
Application number
EP15718982.0A
Other languages
German (de)
French (fr)
Other versions
EP3132034A1 (en
Inventor
Yoshikazu Mikawa
Conrad Lichtenstein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nemesis Bioscience Ltd
Original Assignee
Nemesis Bioscience Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=53008804&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP3132034(B1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority claimed from GBGB1406674.0A external-priority patent/GB201406674D0/en
Priority claimed from GB201413719A external-priority patent/GB201413719D0/en
Priority claimed from GB201418508A external-priority patent/GB201418508D0/en
Priority to EP20190714.4A priority Critical patent/EP3766971A1/en
Priority to SI201531414T priority patent/SI3132034T1/en
Application filed by Nemesis Bioscience Ltd filed Critical Nemesis Bioscience Ltd
Priority to PL15718982T priority patent/PL3132034T3/en
Publication of EP3132034A1 publication Critical patent/EP3132034A1/en
Publication of EP3132034B1 publication Critical patent/EP3132034B1/en
Application granted granted Critical
Revoked legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention relates to recombinant polynucleotides, compositions and methods for interfering with antibiotic resistance genes, and/or replicons carrying such genes, in microorganisms in order to disable antibiotic resistance in the microorganisms.
  • Antibiotics originally isolated from microorganisms such as Streptomyces, are a powerful way to treat infectious disease.
  • AMR anti-microbial resistance
  • One common route to AMR has been the acquisition of resistance genes evolved in the original antibiotic-producing microorganisms, via horizontal transmission on plasmid vectors.
  • Such plasmids have in some instances acquired multiple antibiotic resistance genes carried by transposable elements and integrons.
  • Host-encoded mutations that modify the bacterial protein target or prevent entry of the antibiotic have also occurred.
  • non-antibiotic bactericides have been used.
  • infection by bacteriophage was developed in the 1920's and although largely discontinued with the discovery of antibiotics, has been retained in certain countries.
  • Current approaches use virulent, lytic bacteriophage that kill bacteria, including antibiotic resistant bacteria, but this opens the way for selection of bacterial variants that are resistant to bacteriophage infection.
  • preparations containing a mixture of different strains of bacteriophage are being used.
  • Another disadvantage of the use of such lytic bacteriophage in patients suffering from sepsis is that cell lysis and death by lytic bacteriophage can release endotoxins from the cell into the blood and can cause endotoxin shock.
  • compositions and methods for combating antibiotic resistant microorganisms are required.
  • a delivery vehicle for introducing a polynucleotide into an antibiotic-resistant microorganism
  • the delivery vehicle is selected from the group consisting of a plasmid (such as a conjugative plasmid or other plasmid replicon), a nucleotide vector, linear double-stranded DNA, a non-virulent bacteriophage and a lysogenic bacteriophage, and comprises a recombinant polynucleotide for inactivation of DNA carrying at least one antibiotic resistance gene encoding an enzyme which confers antibiotic resistance to a microrganism
  • the recombinant polynucleotide comprises a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing multiple RNA guide molecules, wherein each RNA guide molecule:
  • CRISPR clustered regularly interspaced short palindromic repeat
  • the delivery vehicle of the invention may further comprise a nucleic acid sequence which encodes the Cas DNA-binding polypeptide or its functional equivalent or its modified version.
  • the Cas DNA-binding polypeptide may be Cas9 or a functional equivalent or a modified version thereof.
  • the delivery vehicle of the invention may comprise a further RNA guide molecule which targets a gene involved in pathogenicity or other aspects of microbial metabolism, for example a gene involved in bacterial metabolism for biofilm production.
  • the modified Cas DNA-binding polypeptide may comprise a recombinase catalytic domain to reseal target DNA sequence.
  • the gene product that prevents direct killing of the microorganism may be a protein encoded by a replicon subject to degradation due to the DSB caused by the Cas DNA-binding polypeptide, or an antitoxin that neutralises the effect of a toxin or killer function carried by a replicon on which the target DNA sequence is located.
  • the CRISPR array nucleic acid sequence may have or transcribe one or more RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of one or more beta-lactamase genes, for example one or more or all of the genes selected from the group consisting of : NDM, VIM, IMP, KPC, OXA, TEM, SHV, CTX, 0KP, LEN, GES, MIR, ACT, ACC, CMY, LAT, and FOX.
  • compositions comprising the delivery vehicle as defined herein.
  • the composition may be a pharmaceutical composition, a non-pathogenic microorganism such as a commensal bacterium for example in a probiotic formulation, or a dietary supplement.
  • the composition may be formulated for topical, enteral or parenteral administration.
  • a further aspect of the invention is a composition as defined herein for use as a medicament.
  • compositions as defined herein for use in the treatment or prevention of an infection caused by an antibiotic-resistant microorganism comprising one or more antibiotic resistance genes targeted by the RNA guide molecules of the recombinant polynucleotide.
  • a method of inactivating antibiotic resistance in an antibiotic-resistant microorganism comprising introducing into the microorganism the delivery vehicle as defined herein.
  • Another aspect of the invention provides a host cell comprising the recombinant polynucleotide as defined herein.
  • the host cell may be a commensal bacterium.
  • the present application discloses a recombinant polynucleotide comprising a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence sufficiently complementary to a target sequence of an antibiotic resistance gene in a microorganism for the antibiotic resistance gene to be inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof, thereby sensitising the microorganism to the antibiotic.
  • CRISPR CRISPR associated
  • a delivery vehicle for introducing a polynucleotide into a microorganism, such as an antibiotic-resistant microorganism
  • the delivery vehicle comprises a recombinant polynucleotide for inactivation of DNA carrying a gene encoding an antibiotic resistance enzyme which confers antibiotic resistance to the microrganism
  • the recombinant polynucleotide comprises a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence sufficiently complementary to a target DNA sequence of the antibiotic resistance gene for the antibiotic resistance gene to be targeted and inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof, thereby sensitising the microorganism to the antibiotic, wherein the delivery vehicle is a non-virulent or a lysogenic bacteriophage.
  • CRISPR CRISPR associated
  • One general aim of the present invention is inactivation of DNA carrying a gene encoding an antibiotic resistance enzyme using a CRISPR/Cas system.
  • An advantage of the invention is that one or more existing antibiotics can be used to treat infectious disease, as microorganisms become re-sensitised to the antibiotics or are prevented from acquiring antibiotic resistance.
  • the target sequence of an antibiotic resistance gene may be a sequence flanking the gene itself which, if disrupted, inactivates the antibiotic resistance gene.
  • the invention may encompass a target sequence in the plasmid.
  • the present invention will not require new drug development and the concomitant regulatory approval required for each new drug. Rather, the invention provides a tool which can be applied to target and inactivate relevant antibiotic resistance genes directly rather than the gene products. For example, a gene encoding an antibiotic resistance enzyme, or a gene encoding a protein regulating the uptake and export of an antibiotic by altering the membrane permeability and efflux pump expression, respectively, can be targeted.
  • the CRISPR/Cas system is an RNA-mediated genome defense pathway that is part of a natural bacterial and archaeal immune system against nucleic acid invaders, analogous to the eukaryotic RNAi pathway (see for example Grissa et al., 2007, BMC Informatics 8: 172 ; Horvath & Barrangou, 2010, Science, 327: 167-170 ; Gasiunas et al., 2012, Proc. Natl Acad. Sci. USA 109: E2579 ; Marraffini & Sontheimer, 2008, Science, 322: 1843-1845 ; Garneau et.al.,2010, Nature 468: 67 ).
  • Natural CRISPR systems contain a combination of Cas genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.
  • Three types (I-III) of CRISPR systems have been identified thus far in a wide range of bacterial and archaeal hosts.
  • Each CRISPR locus is composed of a series of short DNA direct repeats separated by non-repetitive spacer sequences.
  • the spacer sequences in nature, typically originate from foreign genetic elements such as bacteriophage and plasmids.
  • the series of repeats plus non-repetitive spacer sequences is known as a CRISPR array.
  • the CRISPR array is transcribed and hybridised with repeat complementary tracrRNA followed by cleavage within the direct repeats and processed into short mature dual tracrRNA:crRNAs containing individual spacer sequences, which direct Cas nucleases to a target site (also known as a "protospacer").
  • a target site also known as a "protospacer”
  • the Type II CRISPR/Cas9 system carries out a targeted DNA double-strand break ("DSB") in four steps. Firstly, two RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.
  • tracrRNA hybridises to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs (also referred to herein as "RNA guide molecules gRNA" containing individual or monomer spacer sequences.
  • mature crRNAs also referred to herein as "RNA guide molecules gRNA” containing individual or monomer spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 protein in the form of a ribonucleoprotein to the target DNA via base-pairing between the spacer on the crRNA and the target site on the target DNA.
  • Cas9 mediates cleavage of target DNA and creates a DSB.
  • modified CRISPR constructs are used to target antibiotic resistance genes.
  • the recombinant polynucleotide of the invention using such a construct is also referred to herein as an "assassin construct" which is used to effect inactivation of such genes.
  • WO2007/025097 describes the use of CRISPR technology for modulating resistance in a cell against an invading target nucleic acid or a transcription product thereof, especially against invading bacteriophages.
  • Methods for downregulating prokaryotic gene expression using CRISPR technology to target mRNA transcribed by the genes have been suggested for example in WO2010/075424 .
  • WO2012/164565 describes a CRISPR system from Lactoccocus and use of the system for modulating resistance of a cell against an invading target nucleic acid or a transcription product thereof.
  • the present invention concerns inter alia inactivation in an antibiotic-resistant microorganism of genes involved in conferring the antibiotic resistance.
  • the RNA guide molecule mediates binding of the Cas DNA-binding polypeptide or its functional equivalent or its modified version to the antibiotic resistance gene. This mirrors the natural system described above.
  • the Cas DNA-binding polypeptide or its functional equivalent or its modified version may also be capable of binding to RNA or other nucleic acid molecules.
  • the requirement for the Cas DNA-binding polypeptide or its functional equivalent or its modified version to be capable of binding DNA does not exclude the polypepeptide or its functional equivalent or its modified version being capable of binding RNA or other nucleic acid molecules.
  • the Cas DNA-binding polypeptide or its functional equivalent or its modified version may be referred to as a Cas nucleic acid-binding polypeptide or its functional equivalent or its modified version.
  • the microorganism may have a natural endogenous, or introduced engineered, Cas DNA-binding polypeptide or functional equivalent or modified version.
  • the recombinant polynucleotide of the invention is not required to encode the Cas DNA-binding polypeptide or functional equivalent or modified version.
  • the recombinant polynucleotide of the invention may further comprise a nucleic acid sequence which encodes the Cas DNA-binding polypeptide or its functional equivalent or modified version.
  • the recombinant polynucleotide of the invention does not encode the Cas DNA-binding polypeptide or its functional equivalent or modified version but may be used in conjunction with a separate polynucleotide which does.
  • Other means for introducing the Cas DNA-binding polypeptide or its functional equivalent or its modified version into the microorganism may be used.
  • An exemplar Cas DNA-binding polypeptide according to the invention is Cas9 or a functional equivalent thereof or a modified version thereof.
  • the CRISPR array nucleic acid sequence has or transcribes multiple RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of the antibiotic resistance gene or one or more additional antibiotic resistance genes.
  • the or each RNA guide molecule is transcribed from its own promoter sequence.
  • RNA guide molecules allows different antibiotic resistance (or other types of) genes in a microorganism to be targeted and inactivated simultaneously.
  • the recombinant polynucleotide according to various aspects of the invention may additionally be designed to include an RNA guide molecule (such as a further RNA guide molecule) targeting a gene involved in pathogenicity or other aspects of microbial metabolism.
  • an RNA guide molecule such as a further RNA guide molecule
  • certain pathogens form biofilms that make it difficult for antibiotics to gain access to them.
  • One or more genes involved in bacterial metabolism for biofilm production may be targeted.
  • Spacer sequence distal from a promoter are typically less efficiently transcribed. Ideally, multiple RNA guide molecules to different targets should be more or less equally represented. Thus, one promoter transcribing each RNA guide molecule is used (instead of relying on a long polycistronic RNA guide molecule [or precursor crRNA] transcription).
  • bla genes beta-lactamases
  • DNA constructs expressing multiple RNA guide molecules, which are each individually transcribed from their own such promoters, may be used to target a number of different bla genes.
  • the CRISPR array nucleic acid sequence may have or transcribe one or more RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of one or more beta-lactamase genes.
  • the one or more RNA guide molecules may target one or more or all of the genes selected from the group consisting of: NDM, VIM, IMP, KPC, OXA, TEM, SHV, CTX, OKP, LEN, GES, MIR, ACT, ACC, CMY, LAT, and FOX.
  • the one or more RNA guide molecules may comprise a spacer sequence sufficiently complementary to target sequences of the beta lactam family of antibiotic resistance genes, including one or more or all of the following: a first spacer sequence sufficiently complementary to target sequences for NDM-1, -2, -10; a second spacer sufficiently complementary to target sequences for VIM-1, -2, -4, -12, -19, -26, -27- 33, 34; a third spacer sufficiently complementary to target sequences for IMP-32, -38, -48; a fourth spacer sufficiently complementary to target sequences for KPC- 1, -2, -3, -4, -6, -7, -8,-11 , -12 , -14 , -15 , -16, -17; a fifth spacer sufficiently complementary to target sequences for OXA-48; a sixth spacer sufficiently complementary to target sequences for TEM-1,-1B, -3,-139, -162, -183, -192, -197, -198, -209,
  • the antibiotic resistance gene to be inactivated may be located on a chromosome, or on an extrachromosomal replicating DNA molecule known as a replicon and including plasmids and bacteriophage.
  • the CRISPR/Cas system used according to an aspect of the invention generates a DSB in the target sequence.
  • a DSB can lead to degradation and hence loss of the chromosome or replicon suffering such a DSB.
  • the target sequence is located on a bacterial chromosome then the cell may die directly as a consequence of the DSB.
  • some plasmids (including natural plasmids) carry killing functions that only become toxic if the cell loses the plasmid, which is a natural mechanism to ensure faithful inheritance of plasmids in dividing cells.
  • a plasmid carrying the target sequence of the antibiotic resistance gene also carries such a killing function, and the plasmid is lost as a result of the DSB generated, the cell may die (see Sengupta & Austin, 2011, Infect. Immun. 79: 2502-2509 ).
  • the Cas DNA-binding polypeptide may in certain aspects be substituted by a modified Cas DNA-binding polypeptide comprising a recombinase catalytic domain, wherein the modified Cas DNA-binding polypeptide does not generate DSBs but creates a deletion and reseals a site in the target sequence.
  • the modified Cas DNA-binding polypeptide may for example be a modified Cas9 protein comprising a recombinase catalytic domain.
  • the recombinant polynucleotide according to various aspects of the invention may further comprise a nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism, for example thereby increasing the efficiency of delivery of the CRISPR/Cas system to the target microorganism.
  • the gene may confer a growth advantage over non-infected siblings, or genes encoding a bacteriocin - these are protein toxins produced by bacteria to kill or inhibit growth of other bacteria - and corresponding immunity polypeptide may be used.
  • the selective advantage to the microorganism may include or be one which prevents or diminishes the effect of loss of a replicon due to a DSB caused by Cas DNA-binding polypeptide.
  • the nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism may encode an antitoxin that neutralises the effect of a toxin or killer function carried by a replicon on which the target sequence is located.
  • the nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism may encode one or more proteins that are encoded by a replicon subject to degradation due to a DSB caused by Cas DNA-binding polypeptide.
  • a delivery vehicle for introducing a polynucleotide into a microorganism, in which the delivery vehicle comprises the recombinant polynucleotide as defined herein.
  • the delivery vehicle may be a a non-virulent or a lysogenic bacteriophage.
  • the delivery vehicle may be a plasmid such as a conjugative plasmid or other plasmid replicon, a nucleotide vector, or linear double-stranded DNA (dsDNA).
  • the delivery vehicle may be a non-virulent bacteriophage, such as bacteriophage M13, a filamentous phage that infects Escherichia coli cells, replicates and is secreted from the bacterial host cell without killing the bacterial host, which continues to grow and divide more slowly.
  • bacteriophage M13 a non-virulent bacteriophage
  • filamentous phage that infects Escherichia coli cells, replicates and is secreted from the bacterial host cell without killing the bacterial host, which continues to grow and divide more slowly.
  • Another suitable filamentous phage is NgoPhi6 isolated from Neisseria gonorrhoeae that is capable of infecting a variety of Gram negative bacteria without killing the host.
  • lysogenic phage may be used that do not always kills host cells following infection, but instead infect and become dormant prophage.
  • This invention may thus use a novel system to inactivate antibiotic resistance genes in bacteria primarily using bacteriophage that do not kill bacteria, and/or conjugative plasmids and/or direct DNA transformation, as the delivery mechanisms for the recombinant polynucleotide of the invention.
  • composition comprising the delivery vehicle as defined herein.
  • the composition may be a pharmaceutical composition, a non-pathogenic microorganism such as a commensal bacterium for example in a probiotic formulation, or a dietary supplement.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active agents (such the recombinant polynucleotide or the delivery vehicle as described herein) with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism.
  • composition may be formulated for topical, enteral or parenteral administration.
  • compositions of the present invention may, if desired, be presented in a pack, dispenser device or kit, each of which may contain one or more unit dosage forms containing the active agent(s).
  • the pack, dispenser device or kit may be accompanied by instructions for administration.
  • composition as defined herein for use as a medicament.
  • composition as defined herein for use in the treatment or prevention of an infection caused by an antibiotic-resistant microorganism comprising an antibiotic resistance gene targeted by the RNA guide molecule of the recombinant polynucleotide.
  • composition may be administered topically or orally.
  • composition may be administered by intravenous, parenteral, ophthalmic or pulmonary administration.
  • compositions of the present invention for administration topically can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion or dusting powder.
  • compositions provided herein may be formulated for administration by inhalation.
  • the compositions may be in a form as an aerosol, a mist or a powder.
  • compositions described herein may be delivered in the form of an aerosol spray presentation from pressurised packs or a nebuliser, with the use of a suitable propellant such as for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a dosage unit may be determined by providing a valve to deliver a metered amount.
  • the subject may be a plant, a fish, a bird, a reptile or a mammal (such as a human).
  • the delivery vehicle may be transferred from the microorganism directly into another microorganism (such as antibiotic-resistant microorganism) by plasmid conjugation or bacteriophage infection.
  • another microorganism such as antibiotic-resistant microorganism
  • the disclosed method may further comprise a step of simultaneously or subsequently administering to the subject an antibiotic to which a microorganism has become sensitised.
  • a further aspect of the invention provides a method of inactivating antibiotic resistance in a microorganism, the method comprising introducing into the microorganism of the delivery vehicle as defined herein.
  • a host cell comprising the recombinant polynucleotide defined here.
  • the host cell may, for example, be a commensal bacterium.
  • Microorganisms targeted by various aspects of the invention may be on a body surface, localised (for example, contained within an organ, at a site of a surgical wound or other wound, within an abscess), or may be systemic. Included is the treatment of bacterial infections that are amenable to therapy by topical application for example using bacteriophage of the invention.
  • the present disclosure also encompasses coating of surfaces other than body surfaces with the recombinant polynucleotide, delivery vehicle or composition of the present invention, for example wound dressings or medical device surfaces.
  • antibiotic refers to a classical antibiotic that is produced by a microorganism that is antagonistic to the growth of other microorganisms and also encompasses more generally an antimicrobial agent that is capable of killing or inhibiting the growth of a microorganism, including chemically synthesised versions and variants of naturally occurring antibiotics.
  • the term "sufficiently complementary" means that the sequence identity of the spacer sequence and the target sequence is such that the RNA guide molecule comprising the spacer sequence is able to hybridise, preferably specifically and selectively, with the target sequence, thereby allowing for inactivation of the antibiotic resistance gene comprising the target sequence via the CRISPR/Cas system described herein.
  • the spacer sequence may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length with the target sequence.
  • the term "functional equivalent” as used herein refers to a polypeptide which is capable of the same activity as a Cas DNA-binding polypeptide (or, as used herein, a Cas nucleic acid-binding polypeptide).
  • the “functional equivalent” may have the same qualitative biological property as the Cas DNA-binding polypeptide.
  • “Functional equivalents” include, but are not limited to, fragments or derivatives of a native Cas DNA-binding polypeptide and its fragments, provided that the equivalents have a biological activity in common with a corresponding native sequence polypeptide.
  • the functional equivalent may have at least 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length with a Cas DNA-binding polypeptide, for example Cas9 ( Ferretti et al, 2001, PNAS, 98 No. 8: 4658-4663 , Gene ID: 901176, Cas9 GI: 15675041; SEQ ID NO: 99).
  • Cas DNA-binding polypeptide for example Cas9 ( Ferretti et al, 2001, PNAS, 98 No. 8: 4658-4663 , Gene ID: 901176, Cas9 GI: 15675041; SEQ ID NO: 99).
  • Cas DNA-binding polypeptide encompasses a full-length Cas polypeptide, an enzymatically active fragment of a Cas polypeptide, and enzymatically active derivatives of a Cas polypeptide or fragment thereof.
  • Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of a Cas protein or a fragment thereof.
  • modified Cas DNA-binding polypeptide encompasses Cas DNA-binding polypeptides as defined above except that the DSB catalytic function of the polypeptide is replaced by a DNA sealing function due for example to the presence of a recombinase catalytic domain. Further features of such modified Cas DNA-binding polypeptides are described herein.
  • Sequence identity between nucleotide or amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
  • Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat ( Campanella et al., 2003, BMC Bioinformatics 4: 29 ; program available from http://bitincka.com/ledion/matgat), Gap ( Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453 ), FASTA ( Altschul et al., 1990, J. Mol. Biol.
  • sequence comparisons may be undertaken using the "needle" method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score.
  • Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.
  • Default parameters for nucleotide sequence comparisons (“DNA Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: DNAfull.
  • a sequence comparison may be performed over the full length of the reference sequence.
  • the term "gene” refers to a DNA sequence from which a polypeptide is encoded or a non-coding, functional RNA is transcribed.
  • antibiotic resistance gene encompasses a gene, or the encoding portion thereof, which encodes a product or transcribes a functional RNA that confers antibiotic resistance.
  • the antibiotic resistance gene may be a gene or the encoding portion thereof which contributes to any of the four resistance mechanisms described above.
  • the antibiotic resistance gene may for example encode (1) an enzyme which degrades an antibiotic, (2) an enzyme which modifies an antibiotic, (3) a pump such as an efflux pump, or (4) a mutated target which suppresses the effect of the antibiotic.
  • polynucleotide refers to a polymeric form of nucleotide of any length, for example RNA (such as mRNA) or DNA.
  • RNA such as mRNA
  • DNA DNA
  • the term also includes, particularly for oligonucleotide markers, the known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as, for example, those with unchanged linkages, e.g., methyl phosphates, phosphotriesters, phosphoamidates, carbamates, etc. and with charged linkages.
  • polypeptide refers to a polymer of amino acids. The term does not refer to a specific length of the polymer, so peptides, oligopeptides and proteins are included within the definition of polypeptide.
  • polypeptide may include post-expression modifications, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition of "polypeptide” are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids), polypeptides with substituted linkages, as well as other modifications known in the art both naturally occurring and non-naturally occurring.
  • microorganism encompasses prokaryotes such as bacteria and archaea (for example, those belonging to the the Euryarchaeota and Crenarchaeota). Bacteria include both Gram positive and Gram negative bacteria. Some species of clinically significant, pathogenic fungi are included in the definition of microorganisms, for example members of the genus Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys.
  • an advantage of using a bacteriophage or conjugative plasmid that comprises the recombinant polynucleotide of the invention is that each serves as a "Trojan horse” that, following infection of the bacteriophage, or following plasmid conjugation, results in the insertion of the "assassin construct" into the target bacteria or other microorganism.
  • the assassin construct once inserted into the target microorganism also provides an immunisation of the cells against the future arrival of a plasmid harbouring antibiotic resistance genes (see Figure 2 ), in addition, of course to disrupting such genes already present (see Figures 1 and 3 ).
  • the assassin constructs then begin the process of degradation of the antibiotic resistance genes. If a DSB created by the Cas DNA-binding protein of the invention destroys a replicon carrying such an antibiotic resistant gene then a microorganism harbouring the antibiotic resistance gene may be killed directly by an assassin contruct. If the microorganism survives the DSB, the resistance gene will be inactivated, and a patient may then be treated with the antibiotic(s) to which the microorganism has now become sensitised.
  • This present invention provides potential agents for oral, topical and probiotic, dietary supplement delivery as well as an epidemiological tool to silently inactivate antibiotic resistance genes in pathogenic bacteria or other microorganisms (see Figure 3 ).
  • Patients scheduled for surgery, or other treatment in hospital may well be treated with recombinant bacteriophage carrying CRISPR/Cas9 (or other) assassin constructs targeted against antibiotic resistance genes prophylactically in advance of hospital admission. In this way, pathogens present in their microbiome can be directly killed or purged of antibiotic resistance genes in anticipation of any post-operative infection that might occur requiring treatment with antibiotics.
  • this present invention provides an epidemiological tool to silently inactivate antibiotic resistance genes in pathogenic bacteria.
  • a set of CRISPR/Cas9 assassins targeted against selected antibiotics may be constructed.
  • Variables are the bacteriophage (also referred to herein synonymously simply as "phage"), or conjugative plasmid, or DNA, delivery agent: a range of bacteriophage and plasmid agents may be developed that are specific to a range of important bacterial pathogens.
  • a single generalised bacteriophage or plasmid delivery agent needs to be modified to target different bacterial pathogens depends on the details of the specificity of the interactions of either the phage proteins involved in the phage life-cycle, or the plasmid biology and the pathogenic bacterial target.
  • lysogenic phage that infect hosts and become dormant as prophage, as well as non-virulent phage that replicate but do not kill the host, may be developed.
  • lysogenic phage specificity only the lysogenic life cycle of the phage and hence the specificity involved in (i) entry of the phage into the bacterial cell and (ii) its subsequent integration into an attachment site in the target chromosome is required. Integration may not be needed and may be replaced by using the phage to deliver a plasmid that can then excise, for example by cre-lox recombination, and replicate independently in the cell.
  • Non-virulent M13 phage and derivatives thereof may be used.
  • a functional lytic cycle in lysogenic phage may be retained such that low levels of entry into the lytic cycle in lysogenised bacteria will generate new phage that can go on to subsequently infect other bacteria (either pathogenic bacteria or non-pathogenic bacteria, to provide immunity). From the point of view of the epidemiological spread of the phage in the pathogenic population this may not be necessary and a single initial infection may suffice. This can be tested experimentally. Optimal conditions for efficient infection and the appropriate multiplicity of infection are identified.
  • E. coli carrying multiple drug resistance, for example, to ampicillin, chloramphenicol, and kanamycin or targeted against commonly used antibiotics against which resistance is widespread.
  • KPC Klebsiella pneumoniae carbapenemase
  • the phage delivery system (see for example Figure 2 , route 2) may be suitable for the treatment of wounds and burns infected by antibiotic-resistant bacteria.
  • Bacteriophage lambda has a specific integration site in the host chromosome known as the lambda attachment site. Bacteriophage Mu is able to integrate randomly into the host genome and has the advantage that no specific attachment sites in bacteria are required.
  • Phage may also be used to deliver a plasmid replicon containing the recombinant polynucleotide of the invention that excises by cre-lox recombination following infection as discussed above or by use of phage P1 that replicates as an episome.
  • the specificity involved in successful infection is recognition of a membrane protein on the bacterial cell surface. In the case of lambda this is the maltose permease protein that transports the sugar maltose into the bacterial cell. In the case of bacteriophage Mu the receptor is LPS.
  • Male-specific phage M13 that infect E.coli cells carrying the F-factor plasmid may also be used to deliver CRISPR/Cas9 constructs (or other assassin constructs of the invention) targeted against one or more antibiotic resistance genes.
  • M13 is a well studied phage, which replicates, but does not kill the bacterial host.
  • Enterobacteriaceae carrying resistance to the same antibiotics may include Salmonella typhimurium, and Shigella flexneri in addition to Klebsiella pneumoniae discussed above.
  • modified phage are constructed with different genes encoding the tail fibre protein of the bacteriophage in order to allow it to interact with a different receptor present on the bacterial surface. Lysogenic phage, or male-specific phage, like M13, that are natural hosts of these bacteria may be used.
  • phage Mu carries an invertible G segment (regulated by a Mu-encoded site-specific recombinase, Gin) giving rise to two phage types G(+) able to infect E.coli K12 and G(-) able to infect Enterobacter cloacae, Citrobacter freundii Serratia marcescens and Erwinia carotovora.
  • Neisseria gonorrhoeae filamentous phage NgoPhi6, or modified forms thereof is the Neisseria gonorrhoeae filamentous phage NgoPhi6, or modified forms thereof.
  • the natural (wild type) phage has a wide host range for Gram negative bacteria (alpha, beta and gamma proteobacteria) - see Piekarowicz et al. (2014 J. Virol. 88: 1002 - 1010 ).
  • the natural phage is not lytic but lysogenic and has an integration site on the host genome.
  • the ability of the phage to integrate into the host chromosome is removed and the phage is engineered to replicate independently. The substitution of the left integration site L, CTTATAT with L', CATATAT will eliminate the integration event.
  • the M13 ori and M13 gene II can be used to mimic the M13 phage replication.
  • the modification may be kept to a minimum to maintain the ability of the progeny production from the infected bacteria.
  • the maximum packaging size is unknown, a phage DNA of around 12 Kb is experimentally demonstrated as packageable and phage progeny produced from the bacteria infected with this phage are infectable.
  • the length of the phage is around 4 microns, which is 4.4 times longer than M13 phage particle, and indicates the higher packaging capacity of DNA longer than M13 - i.e.
  • the exemplified structure of the phage vector (phagemid) meets the requirements - see Figure 15 .
  • conjugative plasmids Delivery of the assassin constructs targeted against these antibiotics by selected broad-host range conjugative plasmids may be evaluated (see Figure 4 , route 3).
  • the plasmids may be delivered by a benign non-pathogenic host.
  • the application here may be for the Gl tract as a probiotic and may be used prophylactically.
  • the possible lethal effects of DSBs caused by Cas DNA-binding protein are not a concern since the plasmid will not be transferred to the recipient.
  • Delivery by transformation of linear DNA Delivery of the assassin construct targeted against these antibiotics by linear double-stranded DNA transformation (see Figure 4 , route 1) may also be performed.
  • the DNA is delivered via DNA receptor on the surface of the bacteria from the bacterial surrounding environment.
  • Example 1 The aim of Example 1 is to demonstrate a proof-of-concept for resurrection of antibiotic efficacy by introduction of a CRISPR/Cas9 construct in non-pathogenic bacterial strains of Escherichia coli.
  • the Cas9 coding region plus adjacent regulatory regions and DNA encoding the tracrRNA is extracted by PCR, using sequence-specific DNA primers and cloned into a suitable plasmid cloning vector.
  • a unit repeat of CRISPR array comprising the direct repeats flanking a spacer sequence is similarly extracted by PCR and modified to replace the spacer sequence by a cloning site to allow the subsequent introduction of spacer sequences designed to target DNA regions of choice such as antibiotic resistance genes. It is useful to have a positive selection for bacterial transformants carrying the desired recombinants in which such a spacer sequence has been successfully cloned into the cloning site. Appendix 3 gives an example of such a positive selection.
  • an equivalent CRISPR/Cas9 gene targeting construct is obtained from a pCas9 plasmid such as for example available from Addgene: http://www.addgene.org/42876/.
  • This plasmid carries the Cas9 gene plus a DNA sequences encoding tracrRNA and CRISPR array with a unique cloning site in order to introduce the spacer sequence desired to target a given DNA sequence for cleavage by the Cas9 endonuclease.
  • this existing pCas9 plasmid is described below.
  • Example 1 shows that bacteria carrying a beta-lactamase (bla) gene conferring resistance to the beta-lactam antibiotic, ampicillin become sensitive to ampicillin following introduction of a modified CRISPR/Cas9 construct targeted against the bla gene: the CRISPR/Cas9/anti- bla construct.
  • bla beta-lactamase
  • DH5 ⁇ is (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG ⁇ 80d lac Z ⁇ M15 ⁇ ( lacZYA-argF )U169, hsdR17(r K - m K + ), ⁇ -).
  • JM109 is (endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB + ⁇ (lac-proAB) e14- [F' traD36 proAB + lacl q lacZ ⁇ M15]hsdR17(r K - m K + )).
  • pUC18 Ori pMB1
  • pCas9 pACYC184-based vector with Ori p15A, CRISPR locus plus Cas9 gene, CM R
  • pCRISPR Ori pMB1, CRISPR, Kn R
  • pCas9::anti- bla carries resistance to chloramphenicol
  • selection for cells maintaining pCas9::anti- bla is achieved by addition of chloramphenicol to the growth medium.
  • DH5 ⁇ cells with pBR332 or a low copy plasmid are transformed with pCas9 (that is a plasmid in all respects like pCas9::anti- bla but lacking anti- bla which is the spacer sequence targeted against the bla gene: it is predicted that pCas9, in lacking the anti- bla would not be able to attack and inactivate the bla gene.
  • pCas9 is maintained by the presence of chloramphenicol.
  • Beta-lactamase activity can be detected by nitrocefin, which is a chromogenic derivative of cephalosporin, when the beta-lactam ring is hydrolysed, ultraviolet absorption of intact nitrocefin is shifted to around 500 nm, which allows visual detection of the presence of beta lactamase.
  • nitrocefin which is a chromogenic derivative of cephalosporin
  • beta lactamase activity is seen directly challenging the bacteria on the LB plate containing ampicillin.
  • Total bacteria resistant to chloramphenicol is N 0
  • ampicillin resistant bacteria is N a
  • CRISPR/Cas9/anti- bla -mediated inactivation of the bla gene efficiency can be defined by measuring the fraction of the ampicillin sensitive colony,1-Na/N0.
  • Example 1B an M13 phage delivery system is used to introduce CRISPR/Cas9/anti- bla construct into the E coli strain, JM109, expressing beta lactamase gene from a resident plasmid.
  • M13::CRISPR/Cas9/anti- bla phage recombinant is prepared as follows: the CRISPR/Cas9/anti- bla construct is isolated from pCas9::anti- bla by digesting this construct with Sal I and Xba I . The digested fragment size is 5796 bp. The fragment is cloned at Sal I (GTCGAC) and Xba I (TCTAGA) site of M13mp18 RF I. The entire size of the M13mp18 containing CRISPR/Cas9/ anti- bla construct is 13035 bp.
  • This M13 recombinant phage DNA is transformed to E.coli JM109, a bacterial strain carrying the F' plasmid and recombinant M13 phage extruded from the bacteria is purified and used to introduce the CRISPR/Cas9 construct to E.coli JM109 harbouring pBR322.
  • an equivalent M13::CRISPR/Cas9 phage recombinant lacking anti- bl a region is prepared from pCas9 by restriction enzyme Sal I and Xba I and the amplicon is cloned at Sal I and Xba I site of M13mp18 RF I.
  • M13 infects the bacterial cells it does not kill them, but the phage DNA replicates inside the cell and expresses phage genes.
  • M13 phage infection with an M13::CRISPR/Cas9/anti- bla phage recombinant should result in inactivation of the bla gene, in contrast, in a negative control, to infection by an M13::CRISPR/Cas9 phage recombinant lacking anti- bla region. Because M13 phage infection slows down the rate of cell growth, when infected cells grown on a lawn of cells yield turbid plaques of slow growing infected cells.
  • the efficacy of CRISPR/Cas9/anti- bla- mediated inactivation of the bla gene efficiency is calculated by measuring the fraction of white colonies to red red colonies, when plaques are picked and grown as colonies to remove the background lawn. Plaques may also be picked and plated onto LB plate with/without ampicillin to score the ratio of ampicillin sensitive to ampicillin resistant colonies that will result.
  • crRNA CR05 cleaves phosphodiester bond between 762nd base C and 763rd base C
  • CR30 cleaves phosphodiester bond between 198th base G and 199th base A
  • CR70 cleaves phosphodiester bonds between 575th base T and 576th base A
  • CR90 cleaves phosphodiester bonds between 221st base T and 222nd base A on the beta-lactamase gene.
  • each oligo is phosphorylated and ready for cloning at Bsa I sites. Sites of six base cutter restriction endonucleses are underlined, which are useful to screen the recombinants.
  • Cas 9 gene, CRISPR expression locus and tracrRNA (all from S . pyogenes )
  • tracrRNA The reverse complement of tracrRNA is in lowercase, Cas9 coding sequence (SEQ ID NO: 126) is boxed and direct repeat sequence in CRISPR is in bold. Promoter sequences were predicted by neural network algorithm (http://www.fruitfly.org/seq_tools/promoter.html). The two unique sites, Sal I (GTCGAC) and Xba I (TCTAGA) highlighted in bold italicised black are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18. The pACYC184 backbone sequence is italicised.
  • Cas 9 gene, CRISPR expression locus and tracrRNA (all from S . pyogenes )
  • Backbone vector pACYC184 sequence is italicised, sequence positions are numbered from G of EcoRI site underlined.
  • the reverse complement of tracrRNA is in italicised bold located from 1844 to 1929, Cas9 initiation and termination codons are indicated by bold three letters, starting at nucleotide No. 2225 and ending at 6331 followed by leader sequence 6389-6483 indicated by italicised bold letters, first, second and third direct repeat sequences are underlined, between the first and second direct repeat in which spacer cloning region is located.
  • This spacer cloning region contains two inverted Bsa I sites indicated by bold italicised letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3', respectively.
  • Promoter sequences were predicted as above, indicated by lower case for forward promoter for Cas9 and leader sequence and italicised lower case for reverse promoter for tracrRNA, the putative transcription start site is indicated by bold uppercase.
  • the two unique sites, Sa l I (GTCGAC) and Xba I (TCTAGA) highlighted in bold italicised black are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18.
  • Bsa I recognition sites are underlined. lac operator 3 and 1 are bold italicised.
  • Tac promoter is in bold letters, -35 and -10 region in the promoter sequence are underlined, Shine-Dalgarno sequence is boxed, first dipeptide is in bold M and R followed by three consecutive termination codons italicised TAA. Tryptophan terminator signal sequence is employed, indicated by italicised letters.
  • tracrRNA hybridises to the direct repeat region of pre-crRNA indicated by upper case letters using the sequence underlined.
  • Bacterial RNase III cleaves the double-stranded RNA region at indicated position "I", first processing event.
  • the first processing event may also be depicted as follows: tracrRNAs indicated by lower case letters hybridise to the direct repeat region of pre-crRNA indicated by upper case bold black letters. Bacterial RNase III cleaves the double-stranded RNA region at indicated position with arrows, which is first processing event. Phosphodiester bond between 22 nd and 23 rd base in the first and second direct repeat are cleaved. Italicised spacer sequence CR90 is boxed.
  • the 2nd cleavage point (*) is around 20 nt away from the 3' end of the spacer sequence. "Note that the 2nd processing event occurs at a specific distance from the 1st cleavage within the repeats. Considering that spacer sequences are not identical among each other, it is thus likely that the 2nd processing event within the spacers is distance-dependent rather than sequence-dependent" (see Supplementary figure 2 legend in Nature. Mar 31, 2011;471 (7340):602-607 ).
  • the second processing event may also be depicted as follows: The 2nd cleavage point indicated by arrows around 20 nt away from the 3' end of the spacer sequence in the above figure.
  • the part of the target beta-lactamase DNA sequence containing CR90 is shown. crRNA hybridises to its complementary sequence of the target region, Cas9 cleavage points are indicated by dot ".” and the PAM sequence tgg is indicated in the box.
  • the following experiments describe some proof-of-concept experiments performed to demonstrate that the CRISPR-Cas9 system can be used to inactivate antibiotic resistance in bacteria. They describe the construction of a generally applicable DNA cassette, described in the Examples to deliver the CRISPR-Cas9 system plus a derivative carrying a spacer sequence targeted against an antibiotic resistance gene for delivery by naked DNA transformation and bacteriophage infection and also to demonstrate inhibition of the spread of antibiotic resistance by plasmid conjugation.
  • pNB100 is a vector to express the CRISPR-Cas9 system in E.coli with the appropriate unique restriction site, Bsa I, to clone any desired spacer sequence between two direct repeats in the CRISPR locus.
  • the backbone of the vector is derived from pACYC184 and the CRISPR-cas9 locus is inserted into Eco RV site of the vector.
  • Three regions of the CRISPR-cas9 locus were amplified by PCR from the genomic DNA of Streptococcus pyogenes strain SF370, purchased from the ATCC, and assembled by Gibson assembly ( Gibson DG, et.al. Nature Methods 2009; 6: 343-345 ) along with the pACYC184 vector in the reaction.
  • the sequence of the final construct was verified by Sanger sequencing.
  • the CRISPR-Cas9 activity was confirmed using a derivative of pNB100, pNB102, carrying a spacer sequence targeted against the beta lactamase genes of the bacterial transposons Tn3 and Tn1.
  • sequences are the three regions amplified by PCR. Underlined sequences are template-specific primer sequences, bold letters are overlapping sequences used for Gibson assembly.
  • PCR conditions to generate the three fragments were: Fragment 1 Fragment 2 Fragment 3 5X Q5 Reaction Buffer 1 x 1 x 1 x 10 mM dNTPs 200 ⁇ M 200 ⁇ M 200 ⁇ M 10 ⁇ M Forward Primer 0.5 ⁇ M 0.5 ⁇ M 0.5 ⁇ M 10 ⁇ M Reverse Primer 0.5 ⁇ M 0.5 ⁇ M 0.5 ⁇ M S .
  • the total number of nucleotides is 9578 bp.
  • the backbone vector pACYC184 sequence is italicised, sequence positions are numbered from G of Eco RI site (GAATTC) underlined.
  • tracrRNA is located at nucleotide No. from 1889 to 1974 indicated bold letters, Cas9 initiation and termination codons are indicated by bold three letters, starting at nucleotide No. 2270 and ending at 6376 followed by leader sequence 6462-6556 indicated by italicised bold letters, first and second direct repeat sequences are underlined, between which spacer cloning region (30 mer) is located.
  • This spacer cloning region contains two inverted Bsa I sites indicated by bold italicised letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3', respectively and one unique Aat II (5'-GACGTC-3') site also indicated by bold italicised to reduce self-ligation in the event of incomplete Bsa I digestion. Note the transition and transversion base changes G6573A, A6779T that were detected by Sanger sequencing and are shown in bold letters, respectively. However, these point mutations do not affect the CRISPR-Cas9 activity, which will be shown in the later section. The two unique sites, Sal I (GTCGAC) and Xba I (TCTAGA) highlighted in bold italicised letters are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18.
  • a plasmid map of pNB100 is shown in Figure 12 .
  • the desired spacer sequence can be cloned in the clockwise direction between Bsa I sites.
  • This vector contains the p15A origin at 1393-848 and cat (chloramphenicol resistant) gene at 219-9137.
  • Cutting positions of each restriction enzyme, indicated in the parentheses, refer to the position of the 5' cutting sites on the top strand within the recognition sequence.
  • pNB100 was digested with Bsa I and Aat II followed by purification using Agencourt ampure beads.
  • the spacer sequence CR30 was employed from the discussion in B. Selection of spacer sequence from the target sequence in the Materials and Methods section above.
  • the CR30 sequence is as follows :
  • This CR30 cassette was ligated to pNB100 by T4 DNA ligase and transformed to DH5 ⁇ competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the bottom sequence of the CR30 cassette as a reverse primer and a forward primer CF1: 5'-acgttgacgaatcttggagc, which anneals at 6209-6228 region on the recombinant plasmid to generate 409 bp PCR amplicon.
  • PCR positive clones were sequenced to confirm the CR30 spacer sequence and this recombinant clone is designated as pNB102 and used for in vitro beta lactamase- gene disruption experiments.
  • CR30 spacer anneals to the sense strand of beta lactamase- gene and cleaves the phosphodiester bonds between 188th and 189th nucleotide on the sense and antisense strand.
  • pNB102 was digested with unique restriction sites Sal I and Xba I to generate two fragments 6044 bp and 3524 bp. The fragment length was calculated from the 5' end of the restricted sites in the top strand within the restriction recognition sites.
  • the 6044 bp fragments containing CRISPR locus with CR30 spacer sequence in the CRISPR array was separated from the 3524 bp fragment and purified on the preparative 1 % agarose gel.
  • M13 mp18 was digested with Sal I - Xba I , followed by Agencourt ampure purification to remove the six bases Sal I -Xba I fragment from the reaction.
  • Nemesis symbiotics can prevent the spread of antibiotic resistance by inhibiting conjugal transfer of conjugative plasmids carrying antibiotic resistance genes from a donor cell to a recipient cell carrying the Nemesis symbiotics.
  • a recipient cell carrying the Nemesis symbiotics with a donor cell carrying a conjugative plasmid, plus a multicopy mobilisable plasmid carrying the bla gene encoding ampicillin resistance.
  • the conjugative plasmid will transfer itself plus the mobilisable plasmid carrying ampicillin resistance to the recipient.
  • Exconjugants may be selected for resistance to both chloramphenicol present on a non-mobilisable plasmid in the recipient and ampicillin received from the donor.
  • Successful Nemesis symbiotic activity will reduce the efficiency of transfer of ampicillin resistance.
  • the donor strain JA200 (F+ thr-1, leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl) also carrying plasmid pNT3 is described by Saka et al. DNA Research 12, 63-68 (2005 ).
  • the plasmid pNT3 is a mobilisable plasmid carrying the bla gene of Tn1.
  • a single colony of the donor JA200 pNT3 was picked from a Luria broth (LB) plate containing 100 ⁇ g/mL Ampicillin and grown shaking at 37°C overnight in 1mL LB medium with 100 ⁇ g/mL Ampicillin.
  • a single colony each of the recipients, DH5 ⁇ pNB100 and DH5 ⁇ pNB102 was picked from a LB plate containing 35 ⁇ g/mL Chloramphenicol and grown shaking at 37°Covernight in 1mL LB with 35 ⁇ g/mL Chloramphenicol.
  • 50 ⁇ L of cells were added to 1mL LB in Eppendorf tubes and centrifuged 60 sec at 12500 rpm.
  • Cells were resuspended in 50 ⁇ L LB. To set up the mating JA200 pNT3 was spotted onto an LB plate, then 2 ⁇ L of each DH5 ⁇ carrying pNB100 and pNB102 were added to this spot. Separate 2 ⁇ L spottings of donor and recipients were also performed (i.e. not mated). Plates were incubated at 37°C for 4 hours. Cells were removed resuspended in LB and 100 ⁇ L plated on LB plates containing both 100 ⁇ g/mL Ampicillin and 35 ⁇ g/mL Chloramphenicol (LB ApCm).
  • LB ApCm Chloramphenicol
  • Photographs in Figure 13 show platings of the matings between: (A) JA200 pNT3 x DH5 ⁇ pNB100 (as expected lacking Nemesis symbiotic activity); and (B) JA200 pNT3 x DH5 ⁇ pNB102 (showing Nemesis symbiotic activity).
  • the number of cells on LB Cm plus LB Ap plates should equal the number of cells on LB plates.
  • DH5 ⁇ competent cells purchased from New England Biolabs were transformed with pBR322 (carrying the bla gene derived from Tn3) and selected on LB Ap100 plates. Competent cells of the derived strain DH5 ⁇ pBR322 were then prepared using the CaCl2 protocol 25 (1.116) as described by Sambrook and Russell in Molecular Cloning: A Laboratory Manual (3rd Edition, 2001 ) and subsequently transformed with plasmids pNB100, pNB102 and pACYC184 with selection for CmR. Transformant colonies were then picked onto LB Cm35 and LB Ap100 plates. Primary transformants were replica toothpicked onto both LB Cm35 and LB Ap100 plates and incubated overnight at 37°C.
  • Example 3 The aim of Example 3 is to extend the proof-of-concept for resurrection of antibiotic efficacy by introduction of a CRlSPR/Cas9 construct in a pathogenic bacterial strain of Klebsiella pneumoniae.
  • Resistance to a newer class of beta-lactam antibiotics the Carbapenems has emerged in Enterobacteriaceae by the acquisition of new bla genes that encode beta-lactamases able to degrade even the Carbpenems.
  • Klebsiella pneumoniae strains with resistance to Carbapenems, KPC are important causes of morbidity and mortality among hospital-acquired and long-term care-associated infections. Noteworthy is the very high mortality (around 30-70%) among patients with bacteraemia or pulmonary infections.
  • Example 3 shows that a Klebsiella pneumoniae carrying a beta-lactamase ( bla ) gene conferring resistance to the beta-lactam antibiotic, ampicillin become sensitive to ampicillin following introduction of a modified CRISPR/Cas9 construct targeted against the bla gene.
  • the same CRISPR/Cas9/anti- bla construct used in Example 1 or Example 2 is used and is delivered to Klebsiella pneumoniae by conjugation with a donor strain carrying a conjugative plasmid with CRlSPR/Cas9/anti- bla .
  • Ex-conjugant recipient Klebsiella pneumoniae carrying the plasmid with CRlSPR/Cas9/anti- bla. construct is selected for on appropriate media with counter-selection against the donor cell and Klebsiella pneumoniae cells that failed to receive the plasmid.
  • Example 1 and Example 2 efficacy of the CRISPR/Cas9/anti- bla contstruct is evaluated by comparison to a negative control of a Klebsiella pneumoniae ex-conjugant strain that received a plasmid with CRISPR/Cas9 (i.e . lacking the anti- bla region).
  • the beta-lactamase activity can be detected by nitrocefin to count white versus red colonies.
  • beta lactamase activity is seen directly challenging the bacteria on the LB plate with/without ampicillin and measuring the fraction of the ampicillin sensitive colonies versus ampicillin resistant ones.
  • Example 4 provides delivery routes for the therapeutic constructs. These routes all apply to veterinary as well as human applications to be delivered orally, topically, probiotically and for use in surgical irrigation fluids and wound dressings.
  • Phage containing assassin construct as the active ingredient may be administered orally as a stabilised therapeutic preparation: either - administered before antibiotic therapy or administered in the form of an adjuvant complexed to an antibiotic.
  • Conjugative plasmids containing assassin construct as the active ingredient may be administered as a culture of commensal bacteria carrying these plasmids in order to transmit these plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens.
  • Phage containing assassin construct as the active ingredient may be administered topically as a stabilised medication (ointment, spray, powder etc): either - administered before antibiotic topical medication; or administered in the form of a complex with an antibiotic medication.
  • Topical application of conjugative plasmids containing assassin construct as the active ingredient may be via a stabilised culture of commensal bacteria carrying these plasmids in order to transmit the plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens.
  • Phage containing assassin construct as the active ingredient may be added to surgical irrigation fluids and also sprays for disinfecting fomites.
  • Stabilised commensal bacterial cultures containing conjugative plasmids incorporating assassin constructs may be added as coatings to surgical wipes and to wound dressings.
  • a construct is designed to include multiple RNA guide molecules where each RNA guide molecule is transcribed by its own promoter.
  • Figure 16 exemplifies a set of spacer sequences that we have identified encoding 20 guide RNA molecules targeted against 117 different bla genes identified in the NCBI ARDB database for Klebsiella pneumoniae.
  • the beta lactamase gene type, spacer sequence and antibiotic resistance profile in Klebsiella pneumoniae obtained from NCBI ARDB database are shown.
  • Beta lactamase gene sequences were collected from the ARDB database with the keyword Klebsiella pneumoniae. Redundant sequences were removed and unique sequences used for multiple sequence alignment using web program Clustal Omega. One canonical sequence was chosen from each cluster and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder collected. The spacer sequence was chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch. Each of the example spacer sequences shown in the 4 th column has the capability to disrupt the genes in the third column.
  • Beta lactam antibiotics were classified into four classes, penams, cephems, carbapenem and monobactam.
  • One antibiotic name is listed in Figure 16 as an example under each class.
  • the beta lactamase which can open the beta lactam ring is indicated by R.
  • carbapenem is inactivated by KPC.
  • the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG SEQ ID NO: 45
  • the spacer sequence for CMY-a can be employed to LAT-b cleavage.
  • Figure 17 exemplifies a set of spacer sequences encoding 17 guide RNA molecules targeted against 154 different bla genes identified in the CARD database for Klebsiella pneumoniae. Beta lactamase gene type, spacer sequence and antibiotic resistance profile in Klebsiella pneumoniae obtained from the IIDR CARD database are shown.
  • Beta lactamase gene sequences were collected by filtering all the collected beta lactamase genes with the keyword Klebsiella pneumoniae and subjected to multiple sequence alignment using web program Clustal Omega.
  • One canonical sequence from each cluster was chosen and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder collected.
  • the spacer sequence was chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch.
  • the each of the example spacer sequences shown in the 4 th raw has the capability to disrupt the genes in the third column.
  • Beta lactam antibiotics are classified into four classes, penams, cephems, carbapenem and monobactam.
  • penams cephems
  • carbapenem monobactam.
  • the beta lactamase which can open the beta lactam ring is indicated by R.
  • carbapenem is inactivated by KPC.
  • the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG SEQ ID NO: 45
  • a modified Cas DNA-binding polypeptide is created that deletes and reseals rather than leaving a DSB.
  • DSBs caused by Cas DNA-binding polypeptide such as CRISPR-Cas9 can be lethal to the replicon targeted and can result in cell death rather than, for example, re-sensitisation to antibiotics, if an antibiotic resistance gene in the replicon is targeted for inactivation. Immediate cell death rather than such re-sensitisation to antibiotics for subsequent killing by antibiotic may increase selection pressure against delivery of the targeting constructs.
  • DSBs we may modify the Cas9 gene to allow the resealing of the targeted sequence after gene inactivation by introducing a deletion or inversion.
  • Tsai et al. (2014) have constructed a fusion between a catalytically inactive Cas9 (dCas9) protein to the wild-type nuclease domain of the restriction endonuclease Fok1 to increase the specificity of cleavage in eukaryotic cells.
  • Proudfoot et al. (2011) have similarly fused zinc-finger DNA recognition domains to the catalytic domains of recombinases to programme site-specific recombination at designated DNA sequences.
  • the Cas9 resolvase fusion is directed to the desired sites determined by the Cas9 domain:guide RNA spacer sequences; and the fused resolvase is positioned at the recombination site.
  • the fused resolvase is positioned at the recombination site.
  • a and B resolvases must dimerise at each recombination site.
  • two Cas9Rs need to be positioned in close proximity at each recombination site A1A2 and B1B2 as designated by the sequences encoded by the spacers S1-S4.
  • the correct orientations of A1 relative to A2 and B1 relative to B2 will need to be determined experimentally.
  • Figures 19 and 20 show a schematic process as to how this Cas9R fusion resolvase resolves the synapse and parental replicon DNA is recircularised.
  • Figure 21 shows the eight spacer sequences that were designed to target the eight beta-lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48.
  • the primer sequences used in the construction of the plasmids are listed in a table in Figure 22 .
  • plasmid derivative of pNB100 ( Figure 12 ), pNB104A ( Figure 23 ), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against four beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: NDM, IMP, VIM and KPC.
  • plasmid derivative of pNB100, pNB104B ( Figure 24 ), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against four beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: OXA-48, SHV, TEM and CTX-M.
  • a plasmid derivative of pNB100 is constructed where spacer sequences can be expressed from a choice of two different promotors.
  • This plasmid, pNB200 ( Figure 26 ), carries a different unique restriction site downstream of each promotor to clone any desired spacer sequences flanked by their own direct repeats between two direct repeats in the CRISPR locus.
  • Derivatives of pNB200 are described that carry the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against eight beta-lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48.
  • the second promotor is used to express spacers flanked by direct repeats to target the four beta-lactamase families of resistance genes: OXA-48, SHV, TEM and CTX-M.
  • pNB203 Figure 28
  • both promotors are used: the first to express spacers flanked by direct repeats to target four beta-lactamase families of resistance genes: NDM, IMP, VIM and KPC and the second promotor to express spacers flanked by direct repeats to target four beta-lactamase families of resistance genes: OXA-48, SHV, TEM and CTX-M.
  • the tetramer spacer concatemer a+b+c+d shown in Figure 29A was digested with Bsa I , whose restriction site is located in A1 and A2, and ligated to Bsa I spacer cloning sites on pNB100.
  • the structure of the single promoter and spacer region (6221-7001) on pNB104A is shown in Figure 23 .
  • the four-space concatemer contains spacer sequences targeting NDM, IMP, VIM and KPC from the proximal to the distal end of the single promoter.
  • the tetramer spacer sequence concatemer e+f+g+h shown in Figure 29A was digested with Sap I , whose restriction site is located in B1 and B2, and ligated to Sap I spacer cloning sites on pNB200. While screening pNB202 screening this construct was found and confirmed by sequencing. A deletion event between the direct repeats adjacent to leader sequences had occurred and restored the direct repeat sequence to give pNB104B.
  • the single promoter regions (6221-6987) on pNB104B is shown in Figure 24 .
  • the concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located under the single promoter.
  • the concatenated spacer array sequences A and B were amplified from the subcloned vector pCR Blunt II-TOPO_SpacerA and pCR Blunt ll-TOPO_SpacerB with the primer set NB026 and NB029, NB030 and NB033, respectively.
  • At the 3' end of amplicon of spacer A and 5' end of amplicon of spacer B are 20 bases of overlapped sequence from KPC spacer sequence. These two amplicons were gel purified and used for PCR-based pairwise cycle extension reaction in the absence of the primer.
  • the extended material was re-amplified with primer set NB037 (5'-GGGCTGGCAAGCCACGTTTGGTG-3'; SEQ ID NO.
  • the transformants were selected on chloramphenicol LB plates and were screened by PCR with the reverse primer NB021: 5'- GGTGACTGATGGCCACGT (SEQ ID NO: 149) and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC (SEQ ID NO: 148), which anneal at 6368-6385 region and 7203-7225 region, respectively on the recombinant plasmid to generate 858 bp PCR amplicon.
  • PCR-positive clones were sequenced to confirm the eight-spacer concatemer sequence and this recombinant clone is designated as pNB108 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes.
  • a plasmid map of pNB108 is shown in Figure 25 .
  • the plasmid pNB200 contains two identical promotors for gRNA expression and carries a different unique restriction site Bsa I and Sapl downstream of each promotor to clone any desired spacer sequences flanked by their own direct repeats between two direct repeats in the CRISPR locus.
  • the desired structure of the CRISPR array locus in pNB200 consists two sets of cassettes harbouring promotor-leader-direct repeat-spacer cloning region-direct repeat-tail tandemly.
  • the forward primer NB018 anneals at the 5' end of the leader sequence to the promotor in pNB100 and introduces the first spacer cloning region, second direct repeat and tail sequence.
  • the reverse primer NB019 anneals at the 3' end of leader sequence in pNB100 and introduces a third direct repeat, second spacer cloning region. This amplicon is cloned between BsaI sites on pNB100 to give pNB200.
  • the amplified sequence (SEQ ID NO: 146) with primer NB018 and NB019 from pNB100 as template
  • the forward primer NB018 and the reverse primer NB019 are underlined and the initial annealing sites, for the first PCR cycle, are indicated by bold letters and italicised bold letters, respectively.
  • This amplicon was digested with Bbv I (5'-GCAGCN8/N12) to create four bases compatible protruding 5' ends to Bsa I digested pNB100. Bbv I recognition sites are depicted by lower bold cases.
  • the PCR conditions to generate the dual promotor-leader cassette were: Component Nuclease-Free water 41.25 ⁇ L 10 X PCR Buffer 5 ⁇ L 10 mM dNTPs 1 ⁇ L 10 ⁇ M Forward Primer NB018 1 ⁇ L 10 ⁇ M Reverse Primer NB019 1 ⁇ L pNB100 0.5 ⁇ L QIAGEN Hot Start Taq 0.25 ⁇ L
  • the total number of nucleotides of pNB200 is 9919 bp.
  • the sequence in the modified region is shown below, which replaces the small Bsa I fragment in pNB100.
  • the first and the second promotor sequences are underlined. Leader sequences are in bold case.
  • Direct repeats are italicised and the spacer cloning region between direct repeats are indicated by italicised and underlined.
  • the first spacer array cloning region contains two inverted Bsa I sites indicated by bold italicised underlined letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3' on the vector, respectively and one unique Aat II (5'-GACGTC-3') site also indicated by bold italicised underlined lettes to reduce self-ligation in the event of incomplete Bsa I digestion.
  • the second spacer cloning region contains two inverted Sapl sites indicated by bold italicised underlined letters 5'-GAAGAGC-3' and 5'-GCTCTTC-3' for creating 5' three bases protruding spacer cloning sites 5'-GTT-3' and 5'-TTT-3' on the vector, respectively.
  • a plasmid map of pNB200 is shown in Figure 26 .
  • the desired spacer array sequences can be cloned in the clockwise direction between two Bsa I sites and two Sap I sites independently, for each promotor respectively.
  • the plasmid pNB201 contains the dual promotors of pNB200 from which it is derived but a spacer array sequence targeted to OXA-48, SHV, TEM and CTX-M beta lactamase gene families are expressed from the second promotor.
  • pNB202 was digested with Bsa I followed by agarose gel purification.
  • the concatenated spacer array sequence B was generated by PCR-mediated pair-wise oligo nucleotide concatenation.
  • This four-spacer concatemer array B was cloned to the pCR Blunt ll-TOPO vector, purchased from Life Technologies, and the sequence was confirmed.
  • Sapl cuts out spacer array sequence B as a subclone from the TOPO vector and yields 5' protruding three base compatible bases on both ends for the sites created on pNB200 by Sapl digestion.
  • This four-spacer concatemer B cassette was ligated to pNB200 by T4 DNA ligase and transformed to DH5 ⁇ competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the reverse primer NB021: 5'- GGTGACTGATGGCCACGT (SEQ ID NO: 149) and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC (SEQ ID NO: 148), which anneal at 6368-6385 region and 7307-7329 region, respectively on the recombinant plasmid to generate 962 bp PCR amplicon.
  • PCR positive clones were sequenced to confirm the four-spacer concatemer B sequence and this recombinant clone is designated as pNB202 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes.
  • a plasmid map of pNB202 is shown in Figure 27 .
  • the plasmid pNB203 contains the dual promotors of pNB200 but each promotor expresses four spacers.
  • the first promotor expresses spacer sequence targeted to NDM, IMP, VIM and KPC
  • the second promotor expresses OXA, SHV, TEM and CTX-M beta lactamase genes.
  • the plasmid pNB202 was digested with Bsa I followed by agarose gel purification.
  • the concatenated spacer array sequence A was cut out from pCR Blunt ll-TOPO vector harbouring spacer concatemer A with Bsa I .
  • Bsa I cuts out spacer region, which contains 5' protruding four base compatible bases on both ends for the sites created on pNB202 by Bsa I digestion.
  • This four-spacer concatemer A cassette was ligated to pNB202 by T4 DNA ligase and transformed to DH5 ⁇ competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the reverse primer NB021: 5'- GGTGACTGATGGCCACGT and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC , which anneal at 6368-6385 region and 7479-7501 region, respectively on the recombinant plasmid to generate 1134 bp PCR amplicon.
  • PCR positive clones were sequenced to confirm the four-spacer concatemer A and B sequence and this recombinant clone is designated as pNB203 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes.
  • a plasmid map of pNB203 is shown in Figure 28 .
  • Each unit oligo contains the direct repeat flanking the appropriate spacer sequence at each end. Concatenation reactions are performed between pairwise oligos, i.e. the nearest neighbour unit oligos are concatenated first to generate two unit length oligo, then two unit length oligos are concatenated to generate four unit length of oligo etc.
  • FIG. 29 The schematic structure of tetramer and octamer spacer structures are shown in Figure 29 .
  • S spacer
  • R direct repeat
  • a and B contain Bsa I site to create ligation compatible sites for cloning into pNB100 and downstream of the first promotor of pNB200.
  • C and D contain a Sap I site to create ligation a compatible site for cloning downstream of the second promotor of pNB200.
  • Each oligo has overlapped sequence in the 3' and 5' end to anneal to the nearest neighbour oligo except the first and the last oligo.
  • the first and the last oligo have the overlapping sequence to the second and the penultimate oligo in the 5' end only.
  • four oligos are synthesised.
  • oligo No.1 consists spacer 1 and 2 in the 5' and 3' ends.
  • Oligo No.2 contains reverse complement of spacer 2 and 3 in the 3' and 5' ends.
  • Oligo No.3 contains spacer 3 and 4 in the 5' and 3' ends.
  • Oligo No.4 contains reverse complement of spacer 4 in the 3' end.
  • oligo No.2 can link oligo No.1 and oligo No.3, oligo 4 anneals to 3' end of oligo No.3.
  • Oligo No.1 and oligo No. 2, oligo No. 3 and oligo No. 4 are concatenated in a separate tube using the following PCR reaction conditions.
  • NB026 and NB027, NB028 and NB034, NB035 and NB031, NB032 and NB036 are concatenated.
  • Each concatenated product A1, A2, B1 and B2 was gel purified and set up the second concatenation reaction using the purified A1 and A2, B1 and B2 dimer product in the following PCR condition.
  • spacer concatemer A and spacer concatemer B on pCR Blunt II TOPO vector were amplified with primer pairs NB026 and NB029, NB030 and NB033, respectively and amplicons were gel purified. Purified spacer A and B were utilised as a long primer in the following cycle extension reaction.
  • Component A Nuclease-Free water 35.75 ⁇ L 10 X PCR Buffer 5 ⁇ L 10 mM dNTPs 1 ⁇ L QIAGEN Hot Start Taq 0.25 ⁇ L Gel extracted A 4 ⁇ L Gel extracted B 4 ⁇ L
  • Constructs that are able to inactivate target genes, including antibiotic resistant genes, via the CRISPR-Cas system, and which are an aspect of the present invention are also referred to herein as "Nemesis symbiotics".
  • the CRISPR-Cas9 plasmid derivatives, pNB104A, pNB104B, pNB202 and pNB203 all carry spacer insertions targeted against the selected families of beta-lactamase ( bla ) genes described, and so provide exemplars to demonstrate that a single plasmid construct possesses Nemesis symbiotic activity (NSA) and is therefore able to inactivate representative genes from all 8 different families of beta-lactam antibiotics.
  • NSA Nemesis symbiotic activity
  • the plasmid pNB104A (see Fig. 23 ) is tested for its ability to inactivate representative genes members of the NDM, IMP, VIM and KPC families; pNB104B (see Fig. 24 ) and pNB202 (ss Fig. 27 ) are tested for their ability to inactivate representative genes members of the OXA, SHV, TEM and CTX-M families; and pNB108 (see Fig. 25 ) and pNB203 (see Fig. 28 ) are tested for their ability to inactivate representative genes members of the SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA families.
  • the NSA assay described in Example 2 showed that DNA transformation of an E. coli strain, DH5 ⁇ also carrying the TEM-3 beta lactamase gene on the plasmid pBR322, with plasmid pNB102 converts the transformant to ampiciilin sensitivity (ApS).
  • the plasmid pNB102 encodes resistance to chloramphenicol and the DH5 ⁇ (pBR322) transformants now carrying pNB102 were selected on LB Cm plates and then screened for ApS (see Fig. 14 ).
  • the plasmid pNB102 in expressing the CRISPR/Cas9 system with the spacer sequence encoding the gRNA targeting the TEM-3 gene, inactivated the TEM-3 gene.
  • plasmid derivatives of pBR322 are constructed where the TEM-3 is replaced by representative genes from the other 7 different families of beta-lactam antibiotics: SHV, CTX-M, KPC, VIM, IMP, NDM and OXA. Such genes are obtained from suitable bacterial strains carrying such genes. This allows a direct comparison to the proof of concept experiments described in Example 2 in isogenic genetic backgrounds.
  • a set of E . coli and K. pneumoniae strains carrying representative genes from these seven different families of beta-lactam antibiotics were purchased from Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, UK. These are: NCTC13368, a K . pneumoniae strain carrying the SHV-18 gene; NCTC13353 an E. coli strain carrying the CTX-M-15 gene; NCTC13438 a K. pneumoniae strain carrying the KPC-3 gene; NCTC13440 a K. pneumoniae strain carrying the VIM-1 gene; NCTC13476 an E. coli strain carrying an uncharacterised IMP gene; NCTC13443 a K. pneumoniae strain carrying the NDM-1 gene and NCTC13442 a K.
  • pneumoniae strain carrying the OXA-48 gene carrying the OXA-48 gene. All seven genes encode beta lactamases that are also able to degrade and inactivate the penam class of antibiotics (see Fig. 16 , 17 ). All strains were tested and, as expected, found to be resistant to the penam class antibiotic, ampicillin.
  • Beta lactamase coding sequences are amplified from the cell with appropriate forward and reverse primer set shown below: Strain NCTC No. Resistance gene Forward primer 5' to 3' Reverse primer 5' to 3' NBKp001 13443 NDM-1 NBKp002 13442 OXA-48 NBKp003 13368 SHV-18 NBKp004 13440 VIM-1 NBKp005 13438 KPC-3 NBEc018 13476 IMP-4 NBEc019 13353 CTX-M-15
  • Each forward primer contains a 17 base sequence to restore the beta-lactamase promoter on pBR322, and each reverse primer contains Bsa I site (for NDM-1, OXA-48, SHV-18, KPC-3, IMP-4 and CTX-M15) or Fok I site (for VIM-1) to create 5'-ACCG four base protruding 5' end.
  • Bsa I site for NDM-1, OXA-48, SHV-18, KPC-3, IMP-4 and CTX-M15
  • Fok I site for VIM-1
  • the digested amplicons are ready to ligate using T4 ligase between the Sspl and Bsa I sites on the plasmid pBR322 (purchased from New England Biolabs), after removal of the TEM-3 fragment.
  • Ssp I creates a blunt end and Bsa I creates a 5'-CGGT protruding end.
  • the reverse complement of the coding sequences of the each amplicons after restriction digestion are shown below.
  • the 5' protruding end is underlined and 3' end of the promotor sequence is in bold small letters.
  • DH5 ⁇ competent cells purchased from New England Biolabs are transformed with these ligations followed by selection for the desired recombinants on LB Ampicillin (100 ⁇ g/mL) plates.
  • Plasmid DNA samples are isolated from these transformants and submitted to DNA sequence analysis to confirm that the correct sequence for each of the seven different beta lactamases genes is present in each construct giving the plasmids:
  • pBR322 derivative plasmids so derived are named:
  • Nemesis symbiotic activity (NSA) assay by plasmid conjugation to test activity of constructs carrying multiple spacers targeting beta lactamase gene families
  • the plasmid conjugation assay described in Example 2 may also be used to test the Nemesis symbiotic activity of the new CRISPR/Cas9 plasmid constructs carrying spacer sequences targeting multiple families of beta lactamase genes.
  • the assay involves mating a donor cell carrying the beta lactamase gene on a conjugative plasmid, and hence ampicillin resistant, with a recipient cell carrying the Nemesis symbiotic on a non-mobilisable plasmid encoding chloramphenicol resistance. Exconjugants are selected on LB plates containing both 100 ⁇ g/mL ampicillin and 35 ⁇ g/mL chloramphenicol. Successful Nemesis symbiotic activity is seen in a reduction in the efficiency of transfer of the ampicillin resistance gene.
  • Example 2 As donors, the same strain used in Example 2 was used. This strain, JA200 (F+ thr-1, leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl), also carries plasmid pNT3 as described by Saka et al. (DNA Research 12, 63-68, 2005 ).
  • the plasmid pNT3 is a mobilisable plasmid carrying the TEM-1 beta lactamase gene of Tn1. Conjugation of pNT3 and hence transfer of ampicillin resistance is effected by the transfer functions of the co-resident F+ plasmid.
  • the other potential donors are the set of E. coli and K. pneumoniae strains carrying representative genes from these seven different families of beta-lactam antibiotics that were purchased from Culture Collections, Public Health England, as described above. These strains need to be chloramphenicol sensitive in order to allow selection for exconjugants and were tested for growth on LB chloramphenicol 35 ⁇ g/mL plates.
  • pneumoniae strain carrying the OXA-48 gene were all found to be resistant to chloramphenicol, but NCTC13353, an E. coli strain carrying the CTX-M-15 gene, and NCTC13440, a K. pneumoniae strain carrying the VIM-1 gene, were found to be chloramphenicol sensitive and were taken forward to be tested in matings with the recipients.
  • the strains used in Example 2 serve as controls for testing the new plasmid constructs.
  • the negative control is DH5 ⁇ (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG ⁇ 80dlacZ ⁇ M15 ⁇ (lacZYA-argF)U169, hsdR17(rK- mK+), ⁇ with the plasmid pNB100 encoding the CRISPR/Cas9 cassette but no spacer sequence targeting any antibiotic resistance gene; and the positive control is DH5 ⁇ with the plasmid pNB102 encoding the CRISPR/Cas9 cassette as well as the spacer sequence targeting the TEM beta-lactamase family.
  • DH5 ⁇ strains with the plasmids pNB104A, pNB104B and pNB108. These plasmids also encode the CRISPR/Cas9 cassette in addition to the spacer sequences, all driven off one promotor in the following order, proximal to distal from the promotor (pNB104A): NDM-IMP-VIM-KPC; (pNB104B): OXA-48-SHV-TEM-CTX-M (pNB108): NDM-IMP-VIM-KPC-OXA-48-SHV-TEM-CTX-M.
  • colonies of donors were picked from LB Ap100 (ampicillin 100 ⁇ g/mL) and recipients from LB Cm35 (chloramphenicol 35 ⁇ g/mL) plates into 200 ⁇ L of LB and then mixed 100 ⁇ L of recipients with 35 ⁇ L of donors, then 5 ⁇ L of the mixture were spotted onto LB plates and incubated at 37oC for 5 hours to allow mating.
  • LB Ap100 ampicillin 100 ⁇ g/mL
  • LB Cm35 chloramphenicol 35 ⁇ g/mL
  • Figure 30A shows the results from mating the donor carrying the TEM-1 beta lactamase (JA200 (F+ thr-1, leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl)), and designated (5) in the figure, with the recipients DH5 ⁇ pNB100, designated (-), DH5 ⁇ pNB102, designated (+).
  • a loopful of each mating mixture was resuspended in 220 ⁇ L of LB and 200 ⁇ L of the cells were plated on LB Ap100Cm35 plates to select for exconjugants.
  • the mating of the donor carrying the TEM-1 gene with the recipient carrying pNB104 also shows strong inhibition of transfer of the TEM-1 gene and demonstrates that in the pNB104B construct, carrying the four spacers OXA-48-SHV-TEM-CTX-M 4, the TEM spacer is active.
  • Figure 30B shows the results from mating the strain NCTC13440, a K. pneumoniae strain carrying the VIM-1, designated (2), and the strain NCTC13353, an E. coli strain carrying the CTX-M-15 gene, designated (4). Again a loopful of each such mating mixture was resuspended in 220 ⁇ L of LB and 200 ⁇ L of the cells were plated on LB Ap100Cm35 plates to select for exconjugants. The results show that these strains are able to act as donors and transfer the VIM-1 and the CTX-M-15 genes respectively to the DH5 ⁇ pNB100 negative control recipient lacking Nemesis symbiotic activity (see Fig. 30B , top left and top right respectively).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Description

  • The invention relates to recombinant polynucleotides, compositions and methods for interfering with antibiotic resistance genes, and/or replicons carrying such genes, in microorganisms in order to disable antibiotic resistance in the microorganisms.
  • Antibiotics, originally isolated from microorganisms such as Streptomyces, are a powerful way to treat infectious disease. However, very quickly bacteria acquired anti-microbial resistance (AMR) to antibiotics in response to selection pressure. One common route to AMR has been the acquisition of resistance genes evolved in the original antibiotic-producing microorganisms, via horizontal transmission on plasmid vectors. Such plasmids have in some instances acquired multiple antibiotic resistance genes carried by transposable elements and integrons. Host-encoded mutations that modify the bacterial protein target or prevent entry of the antibiotic have also occurred.
  • Resistance to antibiotics by microorganisms such as bacterial pathogens is one of our most serious health threats. Infections from resistant bacteria, for example, are now not uncommon, and some pathogens have even become resistant to multiple types or classes of antibiotics. The loss of effective antibiotics undermines our ability to fight infectious diseases and manage the infectious complications common in vulnerable patients, for example those undergoing chemotherapy for cancer, dialysis for renal failure, and surgery, especially organ transplantation, for which the ability to treat secondary infections is critical.
  • When first-line and second-line treatment options are limited by antibiotic resistance or are unavailable, healthcare providers are forced to use alternative antibiotics that may be more toxic to the patient, more expensive and less effective. Even when alternative treatments exist, patients with resistant infections are often more likely to die, while survivors may have significantly longer hospital stays, delayed recuperation, and long-term disability.
  • Many achievements of modern medicine are put at risk by AMR. Without effective antibiotics for care and prevention of infections, the success of treatments such as organ transplantation, cancer chemotherapy and major surgery would be compromised.
    The growth of global trade and travel allows antibiotic resistant microorganisms to be spread rapidly through humans, other animals, and food.
  • Resistance mechanisms fall into four classes:
    1. (1) enzymes that degrade antibiotics, including beta-lactamases that break the beta-lactam ring of the penicillin family of antibiotics;
    2. (2) enzymes that modify antibiotics include aminoglycoside phosphotransferases that phosphorylate aminoglycoside antibiotics such as kanamycin; chloramphenicol acetyl-transferase (CAT) that acetylate chloramphenicol;
    3. (3) efflux pumps that actively export antibiotics from cytoplasm out of the cell, such as the tetracycline efflux pump that is expressed in the presence of tetracycline, plus other pumps, conferring multidrug resistance, that are capable of exporting a range of antibiotics; and
    4. (4) mutations that change the protein target of the antibiotic such that it is no longer inactivated by it; for example, beta-lactams are bactericidal because they inhibit penicillin-binding proteins (PBPs) that are required for peptidoglycan biosynthesis and bacterial cell wall integrity and PBP mutants with reduced binding to beta-lactams will not be inhibited.
  • Several approaches are currently being used or developed to address the problem of antibiotic resistance, including the following.
  • Firstly, new antibiotics, such as derivatives of existing drugs, have been developed. Fewer new antibiotic drugs have been developed, and many are more toxic so are used in the last resort. Microorganisms have acquired resistance to new antibiotics of both types.
  • Secondly, direct inhibition of resistance enzymes has been attempted. Examples include clavulanic acid, a beta-lactamase inhibitor which is used in combination with amoxycillin, a beta-lactam antibiotic (also called Augmentin). Other beta-lactamase antibiotic inhibitors include the carbapenems. But even here resistance has appeared. Blueberry Therapeutics and Avacta are developing peptide affimers to target mechanisms of resistance. Development of new inhibitors of antibiotic resistance enzymes requires a long pipeline of drug development. An alternative approach has been adopted by Dr Eric Brown at McMaster University, Canada, who is screening known drugs (already approved for use) with unrelated targets, for cryptic activity in inactivating antibody resistance mechanisms.
  • Thirdly, non-antibiotic bactericides have been used. For example, infection by bacteriophage was developed in the 1920's and although largely discontinued with the discovery of antibiotics, has been retained in certain countries. Current approaches use virulent, lytic bacteriophage that kill bacteria, including antibiotic resistant bacteria, but this opens the way for selection of bacterial variants that are resistant to bacteriophage infection. To obviate this, preparations containing a mixture of different strains of bacteriophage are being used. Another disadvantage of the use of such lytic bacteriophage in patients suffering from sepsis is that cell lysis and death by lytic bacteriophage can release endotoxins from the cell into the blood and can cause endotoxin shock.
  • Further compositions and methods for combating antibiotic resistant microorganisms are required.
  • According to an aspect of the present invention, there is provided a delivery vehicle for introducing a polynucleotide into an antibiotic-resistant microorganism, wherein the delivery vehicle is selected from the group consisting of a plasmid (such as a conjugative plasmid or other plasmid replicon), a nucleotide vector, linear double-stranded DNA, a non-virulent bacteriophage and a lysogenic bacteriophage, and comprises a recombinant polynucleotide for inactivation of DNA carrying at least one antibiotic resistance gene encoding an enzyme which confers antibiotic resistance to a microrganism, wherein the recombinant polynucleotide comprises a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing multiple RNA guide molecules, wherein each RNA guide molecule:
    1. (i) is transcribed from its own promoter sequence;
    2. (ii) mediates the binding of a CRISPR associated (Cas) DNA-binding polypeptide or its functional equivalent or its modified version thereof to the at least one antibiotic resistance gene(s); and
    3. (iii) has a spacer sequence sufficiently complementary to a target DNA sequence of the at least one antibiotic resistance gene for the at least one antibiotic resistance gene to be targeted and inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof,
      and wherein:
      • either the Cas DNA-binding polypeptide or a functional equivalent or a modified version thereof is a modified Cas DNA-binding polypeptide which creates a deletion and reseals the target DNA sequence to inactivate the at least one antibiotic resistance gene to prevent double-strand break (DSB)-induced killing of the microorganism, such that the microorganism becomes sensitised to an antibiotic, or
      • the Cas DNA-binding polypeptide or a functional equivalent or a modified version thereof creates a DSB in the target DNA sequence to inactivate the at least one antibiotic resistance gene, and the delivery vehicle further comprises a nucleotide sequence encoding a gene product which prevents direct killing of the microorganism due to the generation of the DSB in the at least one antibiotic resistance gene, such that the microorganism becomes sensitised to an antibiotic.
  • The delivery vehicle of the invention may further comprise a nucleic acid sequence which encodes the Cas DNA-binding polypeptide or its functional equivalent or its modified version.
  • The Cas DNA-binding polypeptide may be Cas9 or a functional equivalent or a modified version thereof.
  • The delivery vehicle of the invention may comprise a further RNA guide molecule which targets a gene involved in pathogenicity or other aspects of microbial metabolism, for example a gene involved in bacterial metabolism for biofilm production.
  • The modified Cas DNA-binding polypeptide may comprise a recombinase catalytic domain to reseal target DNA sequence.
  • The gene product that prevents direct killing of the microorganism may be a protein encoded by a replicon subject to degradation due to the DSB caused by the Cas DNA-binding polypeptide, or an antitoxin that neutralises the effect of a toxin or killer function carried by a replicon on which the target DNA sequence is located.
  • The CRISPR array nucleic acid sequence may have or transcribe one or more RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of one or more beta-lactamase genes, for example one or more or all of the genes selected from the group consisting of : NDM, VIM, IMP, KPC, OXA, TEM, SHV, CTX, 0KP, LEN, GES, MIR, ACT, ACC, CMY, LAT, and FOX.
  • Another aspect of the invention is a composition comprising the delivery vehicle as defined herein.
  • The composition may be a pharmaceutical composition, a non-pathogenic microorganism such as a commensal bacterium for example in a probiotic formulation, or a dietary supplement. The composition may be formulated for topical, enteral or parenteral administration.
  • A further aspect of the invention is a composition as defined herein for use as a medicament.
  • Also provided according to the invention is a composition as defined herein for use in the treatment or prevention of an infection caused by an antibiotic-resistant microorganism comprising one or more antibiotic resistance genes targeted by the RNA guide molecules of the recombinant polynucleotide.
  • Further provided according to the invention a method of inactivating antibiotic resistance in an antibiotic-resistant microorganism, the method comprising introducing into the microorganism the delivery vehicle as defined herein.
  • Another aspect of the invention provides a host cell comprising the recombinant polynucleotide as defined herein. The host cell may be a commensal bacterium.
  • The present application discloses a recombinant polynucleotide comprising a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence sufficiently complementary to a target sequence of an antibiotic resistance gene in a microorganism for the antibiotic resistance gene to be inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof, thereby sensitising the microorganism to the antibiotic.
  • Also disclosed is a delivery vehicle for introducing a polynucleotide into a microorganism, such as an antibiotic-resistant microorganism, in which the delivery vehicle comprises a recombinant polynucleotide for inactivation of DNA carrying a gene encoding an antibiotic resistance enzyme which confers antibiotic resistance to the microrganism, and in which the recombinant polynucleotide comprises a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence sufficiently complementary to a target DNA sequence of the antibiotic resistance gene for the antibiotic resistance gene to be targeted and inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof, thereby sensitising the microorganism to the antibiotic, wherein the delivery vehicle is a non-virulent or a lysogenic bacteriophage.
  • One general aim of the present invention is inactivation of DNA carrying a gene encoding an antibiotic resistance enzyme using a CRISPR/Cas system. An advantage of the invention is that one or more existing antibiotics can be used to treat infectious disease, as microorganisms become re-sensitised to the antibiotics or are prevented from acquiring antibiotic resistance.
  • The target sequence of an antibiotic resistance gene may be a sequence flanking the gene itself which, if disrupted, inactivates the antibiotic resistance gene. For example, if the antibiotic resistance gene is located on a plasmid, the invention may encompass a target sequence in the plasmid.
  • In contrast to prior art approaches of inactivating antibiotic resistance enzymes, the present invention will not require new drug development and the concomitant regulatory approval required for each new drug. Rather, the invention provides a tool which can be applied to target and inactivate relevant antibiotic resistance genes directly rather than the gene products. For example, a gene encoding an antibiotic resistance enzyme, or a gene encoding a protein regulating the uptake and export of an antibiotic by altering the membrane permeability and efflux pump expression, respectively, can be targeted.
  • The CRISPR/Cas system is an RNA-mediated genome defense pathway that is part of a natural bacterial and archaeal immune system against nucleic acid invaders, analogous to the eukaryotic RNAi pathway (see for example Grissa et al., 2007, BMC Informatics 8: 172; Horvath & Barrangou, 2010, Science, 327: 167-170; Gasiunas et al., 2012, Proc. Natl Acad. Sci. USA 109: E2579; Marraffini & Sontheimer, 2008, Science, 322: 1843-1845; Garneau et.al.,2010, Nature 468: 67). Natural CRISPR systems contain a combination of Cas genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPR systems have been identified thus far in a wide range of bacterial and archaeal hosts. Each CRISPR locus is composed of a series of short DNA direct repeats separated by non-repetitive spacer sequences. The spacer sequences, in nature, typically originate from foreign genetic elements such as bacteriophage and plasmids. As used herein, the series of repeats plus non-repetitive spacer sequences is known as a CRISPR array. The CRISPR array is transcribed and hybridised with repeat complementary tracrRNA followed by cleavage within the direct repeats and processed into short mature dual tracrRNA:crRNAs containing individual spacer sequences, which direct Cas nucleases to a target site (also known as a "protospacer"). For example, the Type II CRISPR/Cas9 system, a well-studied example, carries out a targeted DNA double-strand break ("DSB") in four steps. Firstly, two RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Secondly, tracrRNA hybridises to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs (also referred to herein as "RNA guide molecules gRNA" containing individual or monomer spacer sequences. Thirdly, the mature crRNA:tracrRNA complex directs Cas9 protein in the form of a ribonucleoprotein to the target DNA via base-pairing between the spacer on the crRNA and the target site on the target DNA. Finally, Cas9 mediates cleavage of target DNA and creates a DSB.
  • In the present invention, as elaborated herein, modified CRISPR constructs are used to target antibiotic resistance genes. The recombinant polynucleotide of the invention using such a construct is also referred to herein as an "assassin construct" which is used to effect inactivation of such genes.
  • The main focus of using CRISPR technology to date has been for use as a DNA editing tool for reverse genetics, primarily in eukaryotes. However, WO2007/025097 describes the use of CRISPR technology for modulating resistance in a cell against an invading target nucleic acid or a transcription product thereof, especially against invading bacteriophages. Methods for downregulating prokaryotic gene expression using CRISPR technology to target mRNA transcribed by the genes have been suggested for example in WO2010/075424 . WO2012/164565 describes a CRISPR system from Lactoccocus and use of the system for modulating resistance of a cell against an invading target nucleic acid or a transcription product thereof. The present invention, by contrast, concerns inter alia inactivation in an antibiotic-resistant microorganism of genes involved in conferring the antibiotic resistance.
  • According to the invention, the RNA guide molecule mediates binding of the Cas DNA-binding polypeptide or its functional equivalent or its modified version to the antibiotic resistance gene. This mirrors the natural system described above.
  • The Cas DNA-binding polypeptide or its functional equivalent or its modified version may also be capable of binding to RNA or other nucleic acid molecules. In other words, the requirement for the Cas DNA-binding polypeptide or its functional equivalent or its modified version to be capable of binding DNA does not exclude the polypepeptide or its functional equivalent or its modified version being capable of binding RNA or other nucleic acid molecules. The Cas DNA-binding polypeptide or its functional equivalent or its modified version may be referred to as a Cas nucleic acid-binding polypeptide or its functional equivalent or its modified version.
  • For certain applications, the microorganism may have a natural endogenous, or introduced engineered, Cas DNA-binding polypeptide or functional equivalent or modified version. This means that the recombinant polynucleotide of the invention is not required to encode the Cas DNA-binding polypeptide or functional equivalent or modified version. Alternatively, the recombinant polynucleotide of the invention may further comprise a nucleic acid sequence which encodes the Cas DNA-binding polypeptide or its functional equivalent or modified version. In another aspect, the recombinant polynucleotide of the invention does not encode the Cas DNA-binding polypeptide or its functional equivalent or modified version but may be used in conjunction with a separate polynucleotide which does. Other means for introducing the Cas DNA-binding polypeptide or its functional equivalent or its modified version into the microorganism may be used.
  • An exemplar Cas DNA-binding polypeptide according to the invention is Cas9 or a functional equivalent thereof or a modified version thereof.
  • In the recombinant polynucleotide according to various aspects of the invention, the CRISPR array nucleic acid sequence has or transcribes multiple RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of the antibiotic resistance gene or one or more additional antibiotic resistance genes. The or each RNA guide molecule is transcribed from its own promoter sequence.
  • Having multiple RNA guide molecules allows different antibiotic resistance (or other types of) genes in a microorganism to be targeted and inactivated simultaneously.
  • The recombinant polynucleotide according to various aspects of the invention may additionally be designed to include an RNA guide molecule (such as a further RNA guide molecule) targeting a gene involved in pathogenicity or other aspects of microbial metabolism. For example, certain pathogens form biofilms that make it difficult for antibiotics to gain access to them. One or more genes involved in bacterial metabolism for biofilm production may be targeted.
  • Spacer sequence distal from a promoter are typically less efficiently transcribed. Ideally, multiple RNA guide molecules to different targets should be more or less equally represented. Thus, one promoter transcribing each RNA guide molecule is used (instead of relying on a long polycistronic RNA guide molecule [or precursor crRNA] transcription).
  • For example, there are many resistance genes encoding beta-lactamases (bla genes) giving resistance to a large range of different beta-lactam antibiotics. DNA constructs expressing multiple RNA guide molecules, which are each individually transcribed from their own such promoters, may be used to target a number of different bla genes.
  • Thus in aspects of the invention, the CRISPR array nucleic acid sequence may have or transcribe one or more RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of one or more beta-lactamase genes.
  • For example, the one or more RNA guide molecules may target one or more or all of the genes selected from the group consisting of: NDM, VIM, IMP, KPC, OXA, TEM, SHV, CTX, OKP, LEN, GES, MIR, ACT, ACC, CMY, LAT, and FOX.
  • In particular, the one or more RNA guide molecules may comprise a spacer sequence sufficiently complementary to target sequences of the beta lactam family of antibiotic resistance genes, including one or more or all of the following: a first spacer sequence sufficiently complementary to target sequences for NDM-1, -2, -10; a second spacer sufficiently complementary to target sequences for VIM-1, -2, -4, -12, -19, -26, -27- 33, 34; a third spacer sufficiently complementary to target sequences for IMP-32, -38, -48; a fourth spacer sufficiently complementary to target sequences for KPC- 1, -2, -3, -4, -6, -7, -8,-11 , -12 , -14 , -15 , -16, -17; a fifth spacer sufficiently complementary to target sequences for OXA-48; a sixth spacer sufficiently complementary to target sequences for TEM-1,-1B, -3,-139, -162, -183, -192, -197, -198, -209, a seventh spacer sufficiently complementary to target sequences for SHV and its variants; and an eighth spacer sufficiently complementary to target sequences for CTX and its variants.
  • The antibiotic resistance gene to be inactivated may be located on a chromosome, or on an extrachromosomal replicating DNA molecule known as a replicon and including plasmids and bacteriophage.
  • The CRISPR/Cas system used according to an aspect of the invention generates a DSB in the target sequence. Where the target sequence is located on a chromosome or a replicon such as a bacterial chromosome or plasmid, then a DSB can lead to degradation and hence loss of the chromosome or replicon suffering such a DSB. If the target sequence is located on a bacterial chromosome then the cell may die directly as a consequence of the DSB. Additionally, some plasmids (including natural plasmids) carry killing functions that only become toxic if the cell loses the plasmid, which is a natural mechanism to ensure faithful inheritance of plasmids in dividing cells. If a plasmid carrying the target sequence of the antibiotic resistance gene also carries such a killing function, and the plasmid is lost as a result of the DSB generated, the cell may die (see Sengupta & Austin, 2011, Infect. Immun. 79: 2502-2509).
  • In the event that cell death caused by such DSB increases selection pressure for resistance against the recombinant polynucleotide according to various aspects of the present invention, this may be mitigated by, for example, employing a modified Cas DNA-binding polypeptide which seals the target site after generating a deletion to inactivate the target sequence of the antibiotic resistance gene, rather than generate a DSB.
  • Thus the Cas DNA-binding polypeptide according to various aspects of the invention may in certain aspects be substituted by a modified Cas DNA-binding polypeptide comprising a recombinase catalytic domain, wherein the modified Cas DNA-binding polypeptide does not generate DSBs but creates a deletion and reseals a site in the target sequence.
  • The modified Cas DNA-binding polypeptide may for example be a modified Cas9 protein comprising a recombinase catalytic domain.
  • The recombinant polynucleotide according to various aspects of the invention may further comprise a nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism, for example thereby increasing the efficiency of delivery of the CRISPR/Cas system to the target microorganism. For example, the gene may confer a growth advantage over non-infected siblings, or genes encoding a bacteriocin - these are protein toxins produced by bacteria to kill or inhibit growth of other bacteria - and corresponding immunity polypeptide may be used.
  • The selective advantage to the microorganism may include or be one which prevents or diminishes the effect of loss of a replicon due to a DSB caused by Cas DNA-binding polypeptide. For example, the nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism may encode an antitoxin that neutralises the effect of a toxin or killer function carried by a replicon on which the target sequence is located. Also, the nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism may encode one or more proteins that are encoded by a replicon subject to degradation due to a DSB caused by Cas DNA-binding polypeptide.
  • In an aspect of the invention, there is provided a delivery vehicle for introducing a polynucleotide into a microorganism, in which the delivery vehicle comprises the recombinant polynucleotide as defined herein.
  • The delivery vehicle according to various some aspects of the invention may be a a non-virulent or a lysogenic bacteriophage. Alternatively, the delivery vehicle may be a plasmid such as a conjugative plasmid or other plasmid replicon, a nucleotide vector, or linear double-stranded DNA (dsDNA).
  • The delivery vehicle may be a non-virulent bacteriophage, such as bacteriophage M13, a filamentous phage that infects Escherichia coli cells, replicates and is secreted from the bacterial host cell without killing the bacterial host, which continues to grow and divide more slowly. Another suitable filamentous phage is NgoPhi6 isolated from Neisseria gonorrhoeae that is capable of infecting a variety of Gram negative bacteria without killing the host.
  • Alternatively, lysogenic phage may be used that do not always kills host cells following infection, but instead infect and become dormant prophage.
  • This invention may thus use a novel system to inactivate antibiotic resistance genes in bacteria primarily using bacteriophage that do not kill bacteria, and/or conjugative plasmids and/or direct DNA transformation, as the delivery mechanisms for the recombinant polynucleotide of the invention.
  • In a further aspect of the invention, there is provided a composition comprising the delivery vehicle as defined herein.
  • The composition may be a pharmaceutical composition, a non-pathogenic microorganism such as a commensal bacterium for example in a probiotic formulation, or a dietary supplement.
  • As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active agents (such the recombinant polynucleotide or the delivery vehicle as described herein) with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism.
  • The composition may be formulated for topical, enteral or parenteral administration.
  • Compositions of the present invention may, if desired, be presented in a pack, dispenser device or kit, each of which may contain one or more unit dosage forms containing the active agent(s). The pack, dispenser device or kit may be accompanied by instructions for administration.
  • Also provided according to the invention is the composition as defined herein for use as a medicament.
  • In another aspect there is provided according to the invention a composition as defined herein for use in the treatment or prevention of an infection caused by an antibiotic-resistant microorganism comprising an antibiotic resistance gene targeted by the RNA guide molecule of the recombinant polynucleotide.
  • Also disclosed is a method of treating or preventing an infection in a subject caused by an antibiotic-resistant microorganism comprising an antibiotic resistance gene, in which the method comprises the step of introducing into the microorganism a therapeutically effective amount of the composition as defined herein where the RNA guide molecule targets the antibiotic resistance gene, thereby inactivating the antibiotic resistance gene and sensitising the microorganism to the antibiotic.
  • The composition may be administered topically or orally. Alternatively the composition may be administered by intravenous, parenteral, ophthalmic or pulmonary administration.
  • Compositions of the present invention for administration topically can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion or dusting powder.
  • Compositions provided herein may be formulated for administration by inhalation. For example, the compositions may be in a form as an aerosol, a mist or a powder. Thus compositions described herein may be delivered in the form of an aerosol spray presentation from pressurised packs or a nebuliser, with the use of a suitable propellant such as for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Where using a pressurised aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount.
  • In the disclosed method, the subject may be a plant, a fish, a bird, a reptile or a mammal (such as a human).
  • According to the disclosed method, the delivery vehicle may be transferred from the microorganism directly into another microorganism (such as antibiotic-resistant microorganism) by plasmid conjugation or bacteriophage infection.
  • The disclosed method may further comprise a step of simultaneously or subsequently administering to the subject an antibiotic to which a microorganism has become sensitised.
  • A further aspect of the invention provides a method of inactivating antibiotic resistance in a microorganism, the method comprising introducing into the microorganism of the delivery vehicle as defined herein.
  • Also provided according to the invention is a host cell comprising the recombinant polynucleotide defined here. The host cell may, for example, be a commensal bacterium.
  • Microorganisms targeted by various aspects of the invention may be on a body surface, localised (for example, contained within an organ, at a site of a surgical wound or other wound, within an abscess), or may be systemic. Included is the treatment of bacterial infections that are amenable to therapy by topical application for example using bacteriophage of the invention.
  • The present disclosure also encompasses coating of surfaces other than body surfaces with the recombinant polynucleotide, delivery vehicle or composition of the present invention, for example wound dressings or medical device surfaces.
  • Further aspects, features and non-limiting embodiments of the present invention will now be described below with reference to the following drawings:
    • Figure 1 . CRISPR/Cas9-mediated bacterial immunisation against antibiotic resistant genes on a plasmid: I, CRISPR/Cas locus, where boxes denote different spacer sequences targeting different antibiotic resistance genes; II, gRNA-Cas9 where boxes denote different gRNAs targeting each antibiotic resistance gene; III, plasmid harbouring antibiotic resistance gene; IV, target recognition and positioning of Cas9; V, cleaved plasmid, VI: bacteriophage. ApR = ampicillin resistant, CmR = chloramphenicol resistant, KmR = kanamycin resistant. ApS = ampicillin sensitive, CmS = chloramphenicol sensitive, KmS = kanamycin sensitive. Bla= beta-lactamase, CAT = chloramphenicol acetyl transferase, APH = aminoglycoside phosphotransferase. 1. Injection - CRISPR/Cas9 is injected into the bacterial cell along with phage DNA by bacteria-specific phage infection. 2. Lysogenisation - Phage DNA is lysogenised and integrates into the bacterial host chromosome (B. chr.). 3. crRNA biogenesis and assembly of Cas9 - Pre-crRNA is transcribed and hybridised with tracrRNA and processed to make mature crRNA:tracrRNA (an RNA guide molecule, or "gRNA"), which is assembled with Cas9. 4. Recognition - These gRNA-Cas9 complexes recognise target DNA on the plasmid. 5. Cleavage - The gRNA-Cas9 complexes cleave DNA at the sites recognised by crRNAs. 6. Inactivation - This leads to inactivation of the production of antibiotic resistant enzymes; 7. Sensitive - Thus, the bacterial cell becomes sensitive to various antibiotics.
    • Figure 2 . Preventing non-pathogenic bacteria and asymptomatic pathogens from a future encounter with antibiotic resistance genes: I, CRISPR/Cas locus, exemplified here as present in the bacterial chromosome (B. Chr.) and where boxes denote different spacer sequences targeting different antibiotic resistance genes; II, gRNA-Cas9, where boxes denote different gRNAs targeting each antibiotic resistance gene; III, relaxase -an enzyme that makes a strand-specific, sequence-specific nick in double-stranded DNA to initiate conjugal transfer of plasmid DNA; IV, bacteriophage, V, plasmid; VI, double-stranded DNA; VII, single-stranded DNA. Three entry pathways to encounter future antibiotic resistance genes are shown. 1. Transformation- Entry of naked DNA carrying antibiotic resistance gene into the bacterial cell, 2. Bacteriophage-mediated transduction- Phage carrying antibiotic resistance gene infects a bacterial cell. 3 Conjugation- Plasmid carrying antibiotic resistance gene is conjugally transferred from a donor bacterial cell to a recipient bacterial cell. When these antibiotic resistance genes are introduced into the "immunised" cell; it is pre-armed with a CRISPR/Cas locus encoding a gRNA-Cas9 that finds the specific target sequence on the antibiotic resistance gene and cleaves it to disrupt the gene.
    • Figure 3 . Re-sensitising symptomatic pathogens to antibiotics: I, CRISPR/Cas locus in the bacterial chromosome (B. Chr.), where boxes denote different spacer sequences targeting each antibiotic resistance gene; II, gRNA-Cas9, where boxes denote different gRNAs targeting each antibiotic resistance gene; III, antibiotics; IV, protein synthesis is disrupted by cleaving corresponding gene; V, plasmid. This figure shows an asymptomatic pathogen becoming symptomatic because of an opportunistic infection. The pathogen contains plasmid DNA that provides various antibiotic resistance agents such as 1. Efflux pumps- capable of pumping antibiotics out of the bacterial cell, 2 Antibiotic degradation enzymes-and 3. Antibiotic modification enzymes. Each antibiotic resistant agent is disrupted (X) by gRNA-Cas9-mediated site-specific cleavage of corresponding genes.
    • Figure 4 . Three possible CRISPR/Cas delivery routes: I, CRISPR/Cas locus where boxes denote different spacer sequences targeting each antibiotic resistance gene; II, relaxase -an enzyme that makes a strand-specific, sequence-specific nick in double-stranded DNA to initiate conjugal transfer of plasmid DNA; III, bacteriophage; IV, plasmid; V, double-stranded DNA; VI, single-stranded DNA. This figure shows three possible delivery routes of CRISPR/Cas9 expression construct. 1. Transformation- Entry of naked DNA carrying CRISPR/Cas9 into the bacterial cell via a DNA receptor on the cell surface, followed by integration (4) into the bacterial chromosome (B. Chr.), 2. Bacteriophage-mediated transduction- Phage carrying CRISPR/Cas9 infects a bacterial cell, followed by circularisation (5) and then phage-mediated integration (4) into the bacterial chromosome (B. Chr.). 3 Conjugation- Plasmid carrying CRISPR/Cas9 is conjugally transferred from a donor bacterial cell to a recipient bacterial cell, followed by circularisation (5) and plasmid replication. In all cases, boxes in the CRISPR/Cas9 locus denote different spacer sequences targeting each antibiotic resistance gene.
    • Figure 5 . Structure of bidirectional antibiotic resistant transmission model (see also Am J Epidemiol. 2013;178(4):508-520 ) and the effect of antibiotic resistance gene inactivation therapy: A, Antibiotic Resistance is Dominant; B, Antibiotic Sensitivity is Dominant; S, Susceptible, AS, Antibiotic Sensitive; AR, Antibiotic Resistant; T, Transmission; R, Recovery, C, Conversion, CCD, CRISPR/Cas9 delivery. Antibiotic resistance genes in an antibiotic resistant state and the encounter of antibiotic resistance genes in a sensitive state are disrupted for example by gRNA-mediated Cas9 cleavage. Antibiotic resistance gene inactivation therapy converts the bidirectional model (A) to an almost unidirectional conversion model (B) by increasing the conversion rate from antibiotic resistant to sensitive and decreasing the conversion rate from antibiotic sensitive to resistant. The area of each square represents the population density of each state. The width of the arrows is proportional to the magnitude of each parameter (transmission, recovery and conversion), i.e. transmission of antibiotic resistant bacteria is assumed to be higher than antibiotic sensitive bacteria in this figure. Recovery from the infection of pathogens sensitive to antibiotics is higher than for antibiotic resistant pathogens. In the presence of antibiotics, antibiotic-sensitive pathogens are converted to antibiotic resistant pathogens much more than the reverse conversion from resistant to sensitive pathogens because of the selection pressure. The treatment aim is to reverse these conversion parameters to drive the antibiotic resistant pathogen population to a sensitive state by introducing CRISPR/Cas9 into the bacteria.
    • Figure 6 . Structure of superinfection antibiotic resistant transmission model and the effect of antibiotic resistance gene inactivation therapy: The bidirectional antibiotic resistance transmission model depicted in Figure 5 can additionally or alternatively be described by the following superinfection antibiotic resistance transmission model (see also Am J Epidemiol. 2013;178(4):508-520, as for Figure 5). When the antibiotic resistance genes are disrupted for example by gRNA-mediated Cas9 cleavage, this allows the population shift from the antibiotic resistant state to the antibiotic sensitive state via the superinfection state. Disruption of antibiotic resistance genes increases the conversion rate from the antibiotic resistant to the sensitive state. This figure gives a comparison of the relative population density before (Figure 6A) and after (Figure 6B) applying the CRISPR-Cas system. The population is composed of S, Susceptible; Iw, sensitive; Iz, resistant; and Iwz, superinfection state. The area of each circle is proportional to the relative population density in each state. In this simulation, initial density in each state is assumed to be identical (0.25 each, represented by a thin-lined circle). Thick lined circles represent the population density at the equilibrium state. CRISPR-Cas system is clearly contributing to an increase of the population in the susceptible state, thus the recovery rate in the equilibrium state (i.e. S is expanding in B) and to reduce the population in the resistant state (i.e. Iz is shrinking in B). The width of the arrows is proportional to the magnitude of each parameter (infection, recovery and state conversion), i.e. the infection of antibiotic resistant bacteria is assumed to be lower than that of antibiotic sensitive bacteria in this figure. Recovery from the infection of the antibiotic sensitive pathogens is higher than that from the infection of the antibiotic resistant pathogens, because antibiotics are effective on the sensitive strains. In the presence of antibiotics, the antibiotic sensitive pathogens are converted to the antibiotic resistant pathogens much more than the reverse conversion because of the antibiotic selection pressure. The aim of the treatment is to reverse these conversion parameters and to drive the antibiotic resistant pathogen population to the sensitive state.
    • Figure 7 . Structure of CRISPR locus: This figure shows CRISPR locus containing six spacers targeting six different regions. Each crRNA is transcribed monocistronically from the same promoters denoted P to control the transcription level identical for each target. Each crRNA transcript starts with a leader sequence L and terminates with a terminator sequence T. Transcripts of each pre-crRNA are shown as arrows and boxes containing different spacer sequences are indicated by unique shading.
    • Figure 8 . Mapping short bacterial off-target sequence on the bla gene sequence. The figure shows the local alignment of bacterial short sequences (SEQ ID NOs: 3-5, 8-14, 17-23 and 26-33) mapped to the beta lactamase gene. Beta lactamase sequence (SEQ ID NOs: 1-2, 6-7, 15-16 and 24-25) is shown in grey in the top of each panel, PAM (protospacer adjacent motif) sequence is shown in black. Base pairing region with crRNA is underlined, off-target seed sequence on the bacterial genome is italicised. Off-target seed sequence, including PAM sequence, is indicated by two vertical lines. Two numbers on the sequence of beta lactamase gene are the expected two base positions where the phosphodiester bond is cleaved by Cas9 (see Example 1 below).
    • Figure 9 . Predicted crRNA secondary structure. With reference to Figure 8 and Example 1, predicted secondary structures of the crRNA sequences using mFold are shown. The sequences of CR05, CR30, CR70 and CR90 shown in Figure 9 correspond to SEQ ID NOs: 34-37, respectively.
    • Figure 10 . Expected cleavage positions on beta-lactamase gene from pBR322 (SEQ ID NO: 38). Two protospacer sequences, which are base-pairing with crRNA spacer sequences, are underlined (protospacer strand). Two anti-protospacer sequences, which are displaced when protospacer sequences are base pairing with crRNA spacer sequences, are bold italicised (anti-protospacer strand). Associated PAM sequences are indicated either boxed small letters (on protospacer strand) or boxed capital letters (on anti-protospacer strand). Expected cleavage positions are indicated by asterisks. Leader sequence is underlined from 1st to 69th base and highlighted in grey.
    • Figure 11 . Photograph of electrophoretically separated DNA products on a 0.8% agarose gel showing PCR amplicons generated in Example 2 from each of the three regions plus the EcoRV digested vector pACYC184. The marked lanes are as follows: 1*-NEB (N3232S) 1 Kb molecular markers, 2 µg/lane; 2 - Fragment 1, tracrRNA-Cas9 region : 4758 bp; 3* - NEB (N3232S) 1 Kb molecular markers, 0.75 µg/lane; 4 - pACYC184 uncut; 5 - pACYC184 EcoRV cut: 4245 bp; 6 - pACYC184 EcoRV cut: 4245 bp; 7* - NEB (N3232S) 1 Kb molecular markers, 0.12 µg/lane; 8 - Fragment 2, leader and first direct repeat region : 276 bp; 9* - NEB (N3232S) 1 Kb molecular markers, 0.2 µg/lane; and 10 - Fragment 3, Second direct repeat region : 452 bp. Note: * The mass of the each PCR amplicon is estimated by comparing the intensity of the appropriate marker band using NIH ImageJ 1.48v software(http://imagej.nih.gov/ij/).
    • Figure 12 . Plasmid map of pNB100 constructed in Example 2. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). Two direct repeats (DR) are shown as narrow white rectanglular boxes adjacent to the 3' end of leader sequence.
    • Figure 13 . Photographs show results of "Nemesis symbiotic activity" (NSA) according to an embodiment of the invention by bacterial cell mating (see Example 2). The left plate shows JA200 pNT3 x DH5α pNB100 in Ap100Cm35, while the right plate shows JA200 pNT3 x DH5α pNB102 in Ap100Cm35, both plated at 5 x 107 cells /ml.
    • Figure 14 . Photographs show results of NSA according to another embodiment of the invention by plasmid transformation (see Example 2). Left: LB Cm35 plate. Colonies 1-40 are DH5α pBR322 transformed with pNB102; Colonies 45-60 are DH5α pBR322 transformed with pNB100. All colonies show resistance to Cm carried on plasmids pNB100 and pNB102; Right: LB Ap100 plate. Note that colonies 1-40 have lost ApR following transformation with pNB102 carrying the spacer region targeted against the beta-lactamase gene carried on the plasmid pBR322 in strain NBEc001, thereby demonstrating Nemesis symbiotic activity. pNB100 lacking this spacer region but carrying the Cas9 gene is unable to inactivate the beta-lactamase gene.
    • Figure 15 . Shows a phagemid of Ngophi6. ORF11 and ORF7 of Ngophi6 are deleted from Ngophi6 genome. Coding sequences are represented by the arrows indicating the translation polarity of each ORF. The corresponding gene nomencrature of each Ngophi6 phage ORF to M13 are ORF1(gll), ORF2 (gV), ORF4 (gVIII), ORFV (gVIII), ORF8 (gVI), ORF9 (gl). M13 gene nomencretures are in the parenthesis. The second T of the putative left integration site L = CTTATAT is changed to A to give L' = CATATAT to avoid integration. MCS = Multiple cloning site. The location of Ngophi6 and M13mp18 are indicated by the large two open arrows.
    • Figure 16 . Shows a set of spacer sequences (SEQ ID NOs: 39-60) that encode 20 guide RNA molecules targeted against 117 different bla genes identified in the NCBI ARDB database for Klebsiella pneumoniae (see Example 5). Candidate spacer sequences were identified to disrupt all the Klebsiella pneumoniae beta lactamase genes found in the ARDB database. Beta lactamase gene sequences are collected from the ARDB database with the keyword Klebsiella pneumoniae. Redundant sequences were removed and unique sequences used for multiple sequence alignment using web program Clustal Omega. One canonical sequence was chosen from each cluster and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder were collected. The spacer sequence is chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch. Thus each of the example spacer sequences shown in the 4th column has the capability to disrupt the genes in the third column. Beta lactamase genes used in this analysis are: SHV-a = 1, 2, 2a, 5, 5a, 11, 12, 14, 26, 27, 28, 31, 33, 38, 43, 44, 48, 55, 56, 60, 61, 62, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,85, 89, 92, 98, 99, 101, 103, 106, 107, 108, 109, CTXM-b = 1, 3, 10, 12, 15, 22, 32, 54, 60, 62, 68, CTXM-c = 13, 14, 16, 18, 19, 24, 26, CTXM-d = 2, 35, 59, CTXM-e = 26, 63, TEM-f = 1, 1b, 3, ESBL, 139, KPC-g = 1, 2, 3, 4, OKP-h =A11, A12, A16, A17, B13, B-SB613, 6, LEN-i = 2, 17, 18, 19, 20, 21, 22, 24,GES-j = 1, 3, 4, 5, VIM-a = 1, 2, 4, 12, 19, IMP-b = 4, 8, CMY-a= 2, 4, 25, 31, 36, LAT-b = 1, 2, CMY-c = 1, 8b, 10, 19, FOX-d = 1, 5, 7, OXA-a = 1, 30, 47, OXA-2, OXA-9, OXA-b = 10, 17. Beta lactam antibiotics are classified into four classes, penams, cephems, carbapenems and monobactams. One antibiotic name is listed as an example under each class. The beta lactamase, which can open the beta lactam ring is indicated by R. For example, carbapenem is inactivated by KPC. If it is desired to re-sensitise bacteria to carbapenem, the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG should be employed into spacer array to inactivate KPC genes. Note that the spacer sequence for CMY-a can also be employed for LAT-b cleavage. The example of spacer sequences are shown from 5' to 3' direction.
    • Figure 17 . Shows a set of spacer sequences (SEQ ID NOs: 61-77) that encode 17 guide RNA molecules targeted against 154 different bla genes identified in the CARD database for Klebsiella pneumoniae (see Example 5). Candidate spacer sequences were identified to disrupt all the Klebsiella pneumoniae beta lactamase genes found in the CARD database. This table was created with the same method explained in the figure legend in Figure 16. The example of spacer sequences are shown from 5' to 3' direction.
    • Figure 18 . Map of a modified Cas DNA-binding polypeptide, Cas9R. A genetic fusion between Cas9 and Tn3 resolvase. Resolvase and Cas9 are indicated by arrows. The direction of the arrowhead represents the transcription polarity. Functional domain names of Cas9 are shown in the boxes below Cas9 open arrow. This Cas9 is the endonuclease activity deficient mutant dCas9, with amino acid substitutions D10A in RuvCl domain, H840A in HNH domain (as described by Tsai et al. [2014, Nature Biotechnology 32: 569-576]). A mutant Tn3 resolvase (as described by Proudfoot et al. [2011 PLoS ONE 6(4): e19537]) is fused to the N-terminus of this dCas9 via a 12 mer polypeptide linker. Positions of some of these substituted amino acid residues reducing the specificity of the recombination site are indicated by short vertical bars in the N terminal domain, residues 1-148, of the resolvase. The full list of these substitutions is: R2A, E56K, G101S, D102Y, M103I, Q105L, V107F, E132A, L135R. In the Cas9 regions of the fusion: RuvCI, II, III, HNH and PI (PAM interaction) domains are nuclease domains, REC1a and REC1b are recognition domains of repeat and anti-repeat RNA, respectively. REC2 domain does not have any contact to the protospacer-gRNA heteroduplex. Four CRISPR spacer sequences S1, S2, S3 and S4 are arrayed under the expression of one CRISPR leader sequence and are required to bring about the Cas9R-mediated recombination event by the mutant Tn3 resolvase leading to the deletion and re-ligation of the target sequence. Tn3R = Tn3 Resolvase, R = Direct repeat, L = Leader sequence.
    • Figure 19 . Schematic showing site-specific positioning of resolvase by gRNA directed Cas9. The open arrow in step I is the target antibiotic resistance gene on the plasmid for inactivation. Each recombination site A (A1, A2) and B (B1, B2) are recognised by gRNA independently and correctly positioned resolvases are dimerised in close proximity (step II). Dimers in each recombination site A1+A2 and B1+B2 are tetramerised to form a synapse (step III). The synaptic complex (III) is enlarged, gRNAs are presented as dotted arrows designated S1, S2, S3 and S4. Large ovals represent dCas9, longitudinal ovals are resolvases connected via linker peptides. White and grey longitudinal ovals are resolvase catalytic domains dimerising on the recombination site B and A, respectively. The vertical arrows indicate the cleavage position on the recombination sites by resolvase. The thin horizontal parallel arrows represent DNA containing the recombination site A1+A2 and the thick horizontal parallel arrows represent DNA containing the recombination sites B1+B2. The arrowhead shows the 3' end of the DNA sequence. Short black block arrows are locations of each of the PAM sequences.
    • Figure 20 . Schematic showing exchanging half site of the recombination site A1+A2 and B1+B2 followed by strand resolution and sealing break point. Half-site of recombination A1 and B1 are exchanged and ligated and resolved. The region of the target antibiotic gene is resolved as a circular DNA, while the rest of the chromosomal or plasmid replicon is re-circularised (step IV). Short black block arrows are locations of each PAM sequences after resolution.
    • Figure 21 . Shows a set of spacer sequences (SEQ ID NOs: 78-85) that encode 8 guide RNA molecules targeted against the class A genes, SHV-a, CTX-M-b, TEM-c, KPC-d; the class B genes VIM-e, IMP-f, NDM-g and the class D gene, OXA-48 where SHV-a = 1, 1a, 2, 2a, 5, 5a, 11, 12, 14, 18, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 38, 43, 44, 48, 52, 55, 56, 60, 61, 62, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 85, 89, 92, 98, 99, 100, 101, 103, 106, 107, 108, 109, 110, 111, 121, 136, 134, 137, 140, 143, 144, 147, 148, 149, 150, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162, 163, 164, 165, 168, 172, 173, 178, 179; CTXM-b = 1, 3, 10, 12, 15, 19, 22, 32, 52, 54, 59, 60, 62, 68, 71, 80, 81, 99, 141, 147; TEM-c = 1, 1B, 3, 139, 162, 183, 192, 197, 198, 209; KPC-d = 1, 2, 3, 4, 6, 7, 8, 11, 12, 14, 15, 16, 17 ; VIM-e = 1, 2, 4, 19, 26, 27, 33, 34; IMP-f = 4, 8, 32, 38; and NDM-g = 1, 9, 10 (see Example 7).
    • Figure 22 . Shows a table giving the sequences (SEQ ID NOs: 86-98) of the oligonucleotides used in the construction of plasmids pNB200, 202, 203, 104A, 104B and 108 (see Example 7).
    • Figure 23 . Plasmid map of pNB104A constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The tetramer spacer concatemer (Figure 29A) was digested with BsaI, whose restriction site is located in A1 and A2, and ligated to BsaI spacer cloning sites on pNB202 to give pNB203. The single promoter and spacer region (6221-7001) on pNB104A is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against NDM, IMP, VIM and KPC) are located downstream of the single promoter.
    • Figure 24 . Plasmid map of pNB104B constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The single promoter regions (6221-6987) on pNB104B is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located under expression from the single promoter.
    • Figure 25 . Plasmid map of pNB108 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The octamer spacer concatemer (Figure 29B) was digested with BsaI, whose restriction site is located in A1 and A2, and ligated to BsaI spacer cloning sites on pNB100 to give pNB108. The single promoter and spacer region (6221-7225) on pNB108 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against NDM, IMP, VIM, KPC, OXA-48, SHV, TEM and CTX-M) are located under the single promoter.
    • Figure 26 . Plasmid map of pNB200 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The dual promoter cassette was synthesised by PCR from the template pNB100 with primer pair NB018 and NB019, the amplicon was digested with BbvI and ligated to the BsaI site of pNB100 to give pNB200, the small BsaI fragment of pNB100, from position 5776-5741 (see Figure 12) is replaced in the process. The dual promoter and two spacer cloning region (6221-7382) on pNB200 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The restriction enzymes BsaI and SapI are utilised to clone upstream and downstream spacer sequences, respectively.
    • Figure 27 . Plasmid map of pNB202 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The tetramer spacer concatemer (Figure 29A) was digested with SapI, whose restriction site is located in B1 and B2, and ligated to SapI spacer cloning sites on pNB200 to give pNB202. The dual promoter and spacer regions (6221-7329) on pNB202 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located downstream of the second promoter.
    • Figure 28 . Plasmid map of pNB203 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The tetra spacer concatemer a+b+c+d shown in Figure 29A was digested with BsaI, whose restriction site is located in A1 and A2, and ligated to BsaI spacer cloning sites on pNB202 to give pNB203. The dual promoter and spacer regions (6221-7501) on pNB203 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against NDM, IMP, VIM and KPC) are located downstream of the first promoter. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located downstream of the second promoter.
    • Figure 29A . Tetramer spacer concatenation in Example 7. The numbers associating oligos are corresponding to the primer numbers listed in Figure 22. Oligos are pairewise annealed between 26 and 27, 28 and 34, 35 and 31, 32 and 36 via a, c, e and g unique spacer region (I), respectively and extended in individual tubes (II). Dimer concatemer from 26 and 27 concatenate spacer a and b. Dimer concatemer from 28 and 34 concatenate spacer b, c and d. Dimer concatemer from 35 and 31 concatenate spacer e and f. Dimer concatemer from 32 and 36 concatenate spacer f, g and h (II). Concatenated dimmers a+b and b+c+d, e+f and f+g+h are further hybridised via b and f spacer region, respectively and extended to concatenate four spacers a, b, c and d or e, f, g and h (III).
      The tetramer spacer concatemer e+f+g+h was digested with SapI, whose restriction site is located in B1 and B2, and ligated to SapI spacer cloning sites on pNB200 to give pNB202. Tetra spacer concatemer a+b+c+d was digested with BsaI, whose restriction site is located in A1 and A2, and ligated to BsaI spacer cloning sites on pNB202 to give pNB203. a = 20 mer spacer for NDM, b = 20 mer spacer for IMP, c = 20 mer spacer for VIM, d = 20 mer spacer for KPC, e = 20 mer spacer for OXA-48, f = 20 mer spacer for SHV, g = 20 mer spacer for TEM, h = 20 mer spacer for CTX-M.
    • Figure 29B . Octamer spacer concatenation in Example 7. The tetramer spacer concatemer a+b+c+d and e+f+g+h were amplified with primer pair NB026 and NB029, NB030 and NB033, respectively (V), and hybridise tetra concatemer via spacer d region followed by extension to yield octamer spacer a+b+c+d+e+f+g+h. This octamer was digested with BsaI and ligated to BsaI, whose restriction site is located in A1 and A2, sand ligated to BsaI spacer cloning sites on pNB100 to give pNB108. a = 20 mer spacer for NDM, b = 20 mer spacer for IMP, c = 20 mer spacer for VIM, d = 20 mer spacer for KPC, e = 20 mer spacer for OXA-48, f = 20 mer spacer for SHV, g = 20 mer spacer for TEM, h = 20 mer spacer for CTX-M.
    • Figure 30 . Photographs showing results of "Nemesis symbiotic activity" (NSA) according to an embodiment of the invention by bacterial cell mating (see Example 7). Fig 30A, top left plate shows JA200 pNT3 x DH5α pNB100 in Ap100Cm35, while top right plate shows JA200 pNT3 x DH5α pNB102 in Ap100Cm35. Fig 30B shows NCTC13440 x DH5α pNB100, the top left plate and NCTC13353 x DH5α pNB100, the top right plate; and NCTC13440 x DH5α pNB104A the bottom left plate and NCTC13353 x DH5α pNB104B, the bottom right plate all in Ap100Cm35. Fig 30C shows JA200 pNT3 x DH5α pNB100 (5-), NCTC13440 x DH5α pNB100 (2-) and NCTC13353 x DH5α pNB100 (4-), top left plate; and JA200 pNT3 x DH5α pNB108 (5/8), NCTC13440 x DH5α pNB108 (2/8) and NCTC13353 x DH5α pNB108 (4/8), top right plate and JA200 pNT3 x DH5α pNB100 (5+), bottom plate, all in Ap1 00Cm35.
  • As used herein, the term "antibiotic" refers to a classical antibiotic that is produced by a microorganism that is antagonistic to the growth of other microorganisms and also encompasses more generally an antimicrobial agent that is capable of killing or inhibiting the growth of a microorganism, including chemically synthesised versions and variants of naturally occurring antibiotics.
  • The term "sufficiently complementary" means that the sequence identity of the spacer sequence and the target sequence is such that the RNA guide molecule comprising the spacer sequence is able to hybridise, preferably specifically and selectively, with the target sequence, thereby allowing for inactivation of the antibiotic resistance gene comprising the target sequence via the CRISPR/Cas system described herein. For example, the spacer sequence may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length with the target sequence.
  • The term "functional equivalent" as used herein refers to a polypeptide which is capable of the same activity as a Cas DNA-binding polypeptide (or, as used herein, a Cas nucleic acid-binding polypeptide). The "functional equivalent" may have the same qualitative biological property as the Cas DNA-binding polypeptide. "Functional equivalents" include, but are not limited to, fragments or derivatives of a native Cas DNA-binding polypeptide and its fragments, provided that the equivalents have a biological activity in common with a corresponding native sequence polypeptide. Although structural identity is not necessarily required for common biological activity, in one aspect the functional equivalent may have at least 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length with a Cas DNA-binding polypeptide, for example Cas9 (Ferretti et al, 2001, PNAS, 98 No. 8: 4658-4663, Gene ID: 901176, Cas9 GI: 15675041; SEQ ID NO: 99).
  • The term "Cas DNA-binding polypeptide" encompasses a full-length Cas polypeptide, an enzymatically active fragment of a Cas polypeptide, and enzymatically active derivatives of a Cas polypeptide or fragment thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of a Cas protein or a fragment thereof.
  • The term "modified" Cas DNA-binding polypeptide encompasses Cas DNA-binding polypeptides as defined above except that the DSB catalytic function of the polypeptide is replaced by a DNA sealing function due for example to the presence of a recombinase catalytic domain. Further features of such modified Cas DNA-binding polypeptides are described herein.
  • Sequence identity between nucleotide or amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
  • Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; program available from http://bitincka.com/ledion/matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410; program available from http://www.ebi.ac.uk/fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; program available from http://www.ebi.ac.uk/tools/clustalw2) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley; programs available from http://www.ebi.ac.uk/tools/emboss/align). All programs may be run using default parameters.
  • For example, sequence comparisons may be undertaken using the "needle" method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons ("Protein Molecule" option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62. Default parameters for nucleotide sequence comparisons ("DNA Molecule" option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: DNAfull.
  • In one aspect of the invention, a sequence comparison may be performed over the full length of the reference sequence.
  • As used herein, the term "gene" refers to a DNA sequence from which a polypeptide is encoded or a non-coding, functional RNA is transcribed.
  • The term "antibiotic resistance gene" encompasses a gene, or the encoding portion thereof, which encodes a product or transcribes a functional RNA that confers antibiotic resistance. For example, the antibiotic resistance gene may be a gene or the encoding portion thereof which contributes to any of the four resistance mechanisms described above. The antibiotic resistance gene may for example encode (1) an enzyme which degrades an antibiotic, (2) an enzyme which modifies an antibiotic, (3) a pump such as an efflux pump, or (4) a mutated target which suppresses the effect of the antibiotic.
  • The term "polynucleotide" refers to a polymeric form of nucleotide of any length, for example RNA (such as mRNA) or DNA. The term also includes, particularly for oligonucleotide markers, the known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as, for example, those with unchanged linkages, e.g., methyl phosphates, phosphotriesters, phosphoamidates, carbamates, etc. and with charged linkages.
  • The term "polypeptide" as used herein refers to a polymer of amino acids. The term does not refer to a specific length of the polymer, so peptides, oligopeptides and proteins are included within the definition of polypeptide. The term "polypeptide" may include post-expression modifications, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition of "polypeptide" are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids), polypeptides with substituted linkages, as well as other modifications known in the art both naturally occurring and non-naturally occurring.
  • The term "microorganism" encompasses prokaryotes such as bacteria and archaea (for example, those belonging to the the Euryarchaeota and Crenarchaeota). Bacteria include both Gram positive and Gram negative bacteria. Some species of clinically significant, pathogenic fungi are included in the definition of microorganisms, for example members of the genus Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys.
  • Various aspects of the invention are shown in the figures and described below.
  • In the present invention, an advantage of using a bacteriophage or conjugative plasmid that comprises the recombinant polynucleotide of the invention is that each serves as a "Trojan horse" that, following infection of the bacteriophage, or following plasmid conjugation, results in the insertion of the "assassin construct" into the target bacteria or other microorganism.
  • The assassin construct once inserted into the target microorganism also provides an immunisation of the cells against the future arrival of a plasmid harbouring antibiotic resistance genes (see Figure 2), in addition, of course to disrupting such genes already present (see Figures 1 and 3).
  • The assassin constructs then begin the process of degradation of the antibiotic resistance genes. If a DSB created by the Cas DNA-binding protein of the invention destroys a replicon carrying such an antibiotic resistant gene then a microorganism harbouring the antibiotic resistance gene may be killed directly by an assassin contruct. If the microorganism survives the DSB, the resistance gene will be inactivated, and a patient may then be treated with the antibiotic(s) to which the microorganism has now become sensitised.
  • Importantly, there should be no or reduced direct selection pressure acting against this event if and until patients are subsequently treated with antibiotics. Thus there should be little or no direct selection for bacteriophage (or plasmid) resistance in the pathogenic bacteria or other microorganisms and therefore no or less establishment of an "evolutionary arms race" - sometimes a significant limiting feature of the known use of bacteriophage directly as bactericidal agents.
  • In the event that DSB-induced killing of a microorganism increases selection pressure for resistance to a bacteriophage or conjugative plasmid delivery agent, the problem could be mitigated by, for example, using a modified Cas DNA polypeptide as defined herein.
  • This present invention provides potential agents for oral, topical and probiotic, dietary supplement delivery as well as an epidemiological tool to silently inactivate antibiotic resistance genes in pathogenic bacteria or other microorganisms (see Figure 3). Patients scheduled for surgery, or other treatment in hospital, may well be treated with recombinant bacteriophage carrying CRISPR/Cas9 (or other) assassin constructs targeted against antibiotic resistance genes prophylactically in advance of hospital admission. In this way, pathogens present in their microbiome can be directly killed or purged of antibiotic resistance genes in anticipation of any post-operative infection that might occur requiring treatment with antibiotics.
  • Thus this present invention provides an epidemiological tool to silently inactivate antibiotic resistance genes in pathogenic bacteria.
  • To effect exemplification of the invention, a set of CRISPR/Cas9 assassins targeted against selected antibiotics may be constructed. Variables are the bacteriophage (also referred to herein synonymously simply as "phage"), or conjugative plasmid, or DNA, delivery agent: a range of bacteriophage and plasmid agents may be developed that are specific to a range of important bacterial pathogens.
  • The extent to which a single generalised bacteriophage or plasmid delivery agent needs to be modified to target different bacterial pathogens depends on the details of the specificity of the interactions of either the phage proteins involved in the phage life-cycle, or the plasmid biology and the pathogenic bacterial target.
  • Both lysogenic phage that infect hosts and become dormant as prophage, as well as non-virulent phage that replicate but do not kill the host, may be developed. With regards to lysogenic phage specificity, only the lysogenic life cycle of the phage and hence the specificity involved in (i) entry of the phage into the bacterial cell and (ii) its subsequent integration into an attachment site in the target chromosome is required. Integration may not be needed and may be replaced by using the phage to deliver a plasmid that can then excise, for example by cre-lox recombination, and replicate independently in the cell. Non-virulent M13 phage and derivatives thereof may be used.
  • A functional lytic cycle in lysogenic phage may be retained such that low levels of entry into the lytic cycle in lysogenised bacteria will generate new phage that can go on to subsequently infect other bacteria (either pathogenic bacteria or non-pathogenic bacteria, to provide immunity). From the point of view of the epidemiological spread of the phage in the pathogenic population this may not be necessary and a single initial infection may suffice. This can be tested experimentally. Optimal conditions for efficient infection and the appropriate multiplicity of infection are identified.
  • Experiments may be performed in a model system (see Figure 1). For example, E. coli carrying multiple drug resistance, for example, to ampicillin, chloramphenicol, and kanamycin or targeted against commonly used antibiotics against which resistance is widespread.
  • Experiments may also be performed with a target pathogen, for example, Klebsiella pneumoniae carbapenemase ("KPC"). KPC is responsible for infection and death in hospitals in the UK.
  • Delivery by bacteriophage: The phage delivery system (see for example Figure 2, route 2) may be suitable for the treatment of wounds and burns infected by antibiotic-resistant bacteria.
  • Delivery of the assassin construct targeted against these antibiotics by two different E.coli lysogenic bacteriophage: bacteriophage lambda and bacteriophage Mu may be used. Bacteriophage lambda has a specific integration site in the host chromosome known as the lambda attachment site. Bacteriophage Mu is able to integrate randomly into the host genome and has the advantage that no specific attachment sites in bacteria are required.
  • Phage may also be used to deliver a plasmid replicon containing the recombinant polynucleotide of the invention that excises by cre-lox recombination following infection as discussed above or by use of phage P1 that replicates as an episome. The specificity involved in successful infection is recognition of a membrane protein on the bacterial cell surface. In the case of lambda this is the maltose permease protein that transports the sugar maltose into the bacterial cell. In the case of bacteriophage Mu the receptor is LPS.
  • Male-specific phage M13 that infect E.coli cells carrying the F-factor plasmid may also be used to deliver CRISPR/Cas9 constructs (or other assassin constructs of the invention) targeted against one or more antibiotic resistance genes. M13 is a well studied phage, which replicates, but does not kill the bacterial host.
  • Successful elimination of resistance to target antibiotics following the lyogenisation by recombinant bacteriophage, or infection by M13 recombinants carrying the assassin construct directed against these antibiotic resistance genes is evaluated.
  • Studies on other closely related Enterobacteriaceae carrying resistance to the same antibiotics may be performed. These may include Salmonella typhimurium, and Shigella flexneri in addition to Klebsiella pneumoniae discussed above.
  • In order to do this, modified phage are constructed with different genes encoding the tail fibre protein of the bacteriophage in order to allow it to interact with a different receptor present on the bacterial surface. Lysogenic phage, or male-specific phage, like M13, that are natural hosts of these bacteria may be used.
  • The phenomenon of phase variation whereby bacteriophage are able to switch their host specificity by the inversion, at a low frequency, of a DNA segment to control gene expression and express alternative tail fibre proteins may also be exploited. And in that way develop phage delivery systems that are versatile. For example phage Mu carries an invertible G segment (regulated by a Mu-encoded site-specific recombinase, Gin) giving rise to two phage types G(+) able to infect E.coli K12 and G(-) able to infect Enterobacter cloacae, Citrobacter freundii Serratia marcescens and Erwinia carotovora.
  • Another example is the Neisseria gonorrhoeae filamentous phage NgoPhi6, or modified forms thereof. The natural (wild type) phage has a wide host range for Gram negative bacteria (alpha, beta and gamma proteobacteria) - see Piekarowicz et al. (2014 J. Virol. 88: 1002 - 1010). The natural phage is not lytic but lysogenic and has an integration site on the host genome. In one aspect of the invention, the ability of the phage to integrate into the host chromosome is removed and the phage is engineered to replicate independently. The substitution of the left integration site L, CTTATAT with L', CATATAT will eliminate the integration event. In order to replicate the phage genome extra-chromosomally, the M13 ori and M13 gene II can be used to mimic the M13 phage replication. The modification may be kept to a minimum to maintain the ability of the progeny production from the infected bacteria. Although the maximum packaging size is unknown, a phage DNA of around 12 Kb is experimentally demonstrated as packageable and phage progeny produced from the bacteria infected with this phage are infectable. The length of the phage is around 4 microns, which is 4.4 times longer than M13 phage particle, and indicates the higher packaging capacity of DNA longer than M13 - i.e. assuming that the packaging size of DNA is proportional to the size of the phage particle length, DNA packaging capacity of the NgoPhi6 phage will 4 microns/0.9 microns x 6407 nt = 28475 nt, large enough for packaging phage genome along with a recombinant polynucleotide of the invention, including for example a CRISPR-Cas9 construct if required. The exemplified structure of the phage vector (phagemid) meets the requirements - see Figure 15.
  • Delivery by conjugative plasmids: Delivery of the assassin constructs targeted against these antibiotics by selected broad-host range conjugative plasmids may be evaluated (see Figure 4, route 3). Here, the plasmids may be delivered by a benign non-pathogenic host. The application here may be for the Gl tract as a probiotic and may be used prophylactically. In this aspect, the possible lethal effects of DSBs caused by Cas DNA-binding protein are not a concern since the plasmid will not be transferred to the recipient.
  • Similarly demonstration of the successful elimination of resistance to the three target antibiotics following conjugal transfer of plasmids carrying the assassin cargo may be evaluated.
  • Delivery by transformation of linear DNA: Delivery of the assassin construct targeted against these antibiotics by linear double-stranded DNA transformation (see Figure 4, route 1) may also be performed. Here, the DNA is delivered via DNA receptor on the surface of the bacteria from the bacterial surrounding environment.
    • Examples
  • The present invention is illustrated by the following non-limiting examples.
    • Example 1
  • The aim of Example 1 is to demonstrate a proof-of-concept for resurrection of antibiotic efficacy by introduction of a CRISPR/Cas9 construct in non-pathogenic bacterial strains of Escherichia coli.
  • A CRlSPR/Cas9 construct is made directly from genomic DNA isolated from the Streptococcus pyogenes strain SF370, which is obtained from: http://www,straininfo.net/strains/1178GG/browser;jsessionid=A07A638D6D2472EA2FEDBD3 A1928F347.straininfo2. Here the Cas9 coding region plus adjacent regulatory regions and DNA encoding the tracrRNA is extracted by PCR, using sequence-specific DNA primers and cloned into a suitable plasmid cloning vector. A unit repeat of CRISPR array comprising the direct repeats flanking a spacer sequence is similarly extracted by PCR and modified to replace the spacer sequence by a cloning site to allow the subsequent introduction of spacer sequences designed to target DNA regions of choice such as antibiotic resistance genes. It is useful to have a positive selection for bacterial transformants carrying the desired recombinants in which such a spacer sequence has been successfully cloned into the cloning site. Appendix 3 gives an example of such a positive selection.
  • Alternatively an equivalent CRISPR/Cas9 gene targeting construct, though lacking a positive selection for recombinants, is obtained from a pCas9 plasmid such as for example available from Addgene: http://www.addgene.org/42876/. This plasmid carries the Cas9 gene plus a DNA sequences encoding tracrRNA and CRISPR array with a unique cloning site in order to introduce the spacer sequence desired to target a given DNA sequence for cleavage by the Cas9 endonuclease. For exemplification purposes, use of this existing pCas9 plasmid is described below.
  • Example 1 shows that bacteria carrying a beta-lactamase (bla) gene conferring resistance to the beta-lactam antibiotic, ampicillin become sensitive to ampicillin following introduction of a modified CRISPR/Cas9 construct targeted against the bla gene: the CRISPR/Cas9/anti-bla construct.
    • Materials and Methods A. Bacterial strains, plasmids and phage 1. Bacterial strains:
  • DH5α is (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK - mK +),λ-).
    JM109 is (endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36 proAB+ laclq lacZΔM15]hsdR17(rK -mK +)).
    • 2. Plasmids:
  • pUC18 (Ori pMB1); pCas9 (pACYC184-based vector with Ori p15A, CRISPR locus plus Cas9 gene, CMR); pCRISPR (Ori pMB1, CRISPR, KnR).
    • 3. Phage: M13mp18
  • In Example 1A, in one strain E.coli DH5α and carrying pBR322 a medium copy plasmid or alternatively a low copy plasmid, the CRISPR/Cas9/anti-bla construct is delivered, by naked DNA transformation in plasmid pCas9, designated pCas9::anti-bla. The already present plasmid pBR322, or the low copy plasmid expresses the beta-lactamase derived from bacterial transposon Tn3; pBR322 also carries resistance to tetracycline. As pCas9::anti-bla carries resistance to chloramphenicol, selection for cells maintaining pCas9::anti-bla is achieved by addition of chloramphenicol to the growth medium. In a separate negative control experiment DH5α cells with pBR332, or a low copy plasmid are transformed with pCas9 (that is a plasmid in all respects like pCas9::anti-bla but lacking anti-bla which is the spacer sequence targeted against the bla gene: it is predicted that pCas9, in lacking the anti-bla would not be able to attack and inactivate the bla gene. Again pCas9 is maintained by the presence of chloramphenicol.
  • Beta-lactamase activity can be detected by nitrocefin, which is a chromogenic derivative of cephalosporin, when the beta-lactam ring is hydrolysed, ultraviolet absorption of intact nitrocefin is shifted to around 500 nm, which allows visual detection of the presence of beta lactamase.
  • Let the total number of bacteria resistant to chloramphenicol be N0 and the number of beta-lactamase resistant bacteria be Nr. Growth of Nr manifest as red colonies in the presence of nitrocefin. Let the number of beta lactamase sensitive bacteria be Nw. Growth of Nw colonies manifest as white colonies in the presence of nitrocefin. Thus the efficacy of CRISPR/Cas9/anti-bla-mediated inactivation of the blagene efficiency is calculated by measuring the fraction of white colonies, i.e.Nw/N0=(N0-Nr)/N0.
  • Alternatively beta lactamase activity is seen directly challenging the bacteria on the LB plate containing ampicillin. Total bacteria resistant to chloramphenicol is N0, ampicillin resistant bacteria is Na, then CRISPR/Cas9/anti-bla-mediated inactivation of the bla gene efficiency can be defined by measuring the fraction of the ampicillin sensitive colony,1-Na/N0.
  • In Example 1B, an M13 phage delivery system is used to introduce CRISPR/Cas9/anti-bla construct into the E coli strain, JM109, expressing beta lactamase gene from a resident plasmid.
  • M13::CRISPR/Cas9/anti-bla phage recombinant is prepared as follows: the CRISPR/Cas9/anti-bla construct is isolated from pCas9::anti-bla by digesting this construct with SalI and XbaI. The digested fragment size is 5796 bp. The fragment is cloned at SalI (GTCGAC) and XbaI (TCTAGA) site of M13mp18 RF I. The entire size of the M13mp18 containing CRISPR/Cas9/ anti-bla construct is 13035 bp. This M13 recombinant phage DNA is transformed to E.coli JM109, a bacterial strain carrying the F' plasmid and recombinant M13 phage extruded from the bacteria is purified and used to introduce the CRISPR/Cas9 construct to E.coli JM109 harbouring pBR322. As a negative control, an equivalent M13::CRISPR/Cas9 phage recombinant lacking anti-bla region is prepared from pCas9 by restriction enzyme SalI and XbaI and the amplicon is cloned at SalI and XbaI site of M13mp18 RF I.
  • When M13 infects the bacterial cells, it does not kill them, but the phage DNA replicates inside the cell and expresses phage genes. Thus M13 phage infection with an M13::CRISPR/Cas9/anti-bla phage recombinant should result in inactivation of the bla gene, in contrast, in a negative control, to infection by an M13::CRISPR/Cas9 phage recombinant lacking anti-bla region. Because M13 phage infection slows down the rate of cell growth, when infected cells grown on a lawn of cells yield turbid plaques of slow growing infected cells. If nitrocefin is added to the growth medium, the efficacy of CRISPR/Cas9/anti-bla-mediated inactivation of the bla gene efficiency is calculated by measuring the fraction of white colonies to red red colonies, when plaques are picked and grown as colonies to remove the background lawn. Plaques may also be picked and plated onto LB plate with/without ampicillin to score the ratio of ampicillin sensitive to ampicillin resistant colonies that will result.
    • B. Selection of spacer sequence from the target sequence
  • We can use Guide RNA Target Design Tool (see; https://wwws.blueheronbio.com/external/tools/gRNASrc.jsp) from BlueHeron to select spacer sequence from the target. This program simply returns the 20 nt spacer sequence with the appropriate PAM (protospacer adjacent motif) sequence in the 3' end and GC content. It does not consider secondary structure stability and sequence specificity. Secondary structure prediction and specificity search is performed manually.
  • We choose the actual spacer sequence from the candidate sequences obtained in the above program, which should meet the following two criteria: 1) low tendency to form stable secondary structure of crRNA, 2) no target DNA on the host genomic DNA. It may be very difficult to find a unique sequence to satisfy criterion No.2. Considering mismatched target data from Figure 3 E in Jinek et al Science 337, 816 (2012), criterion No.2 is relaxed to allow a matched sequence up to the 12th nucleotide position in the target sequence (the first nucleotide position is counted from just next to the PAM sequence). In other words, when the first 12 mer protospacer sequence of the target sequence is completely matched to the 12 mer sequence of crRNA spacer sequence in the 3' end, but the rest of the sequence is not matched, it is assumed that target dsDNA is not cleaved. The specificity check of the protospacer sequence along the E.coli K12 genome sequence is performed by BLAST. The bla sequence is searched against the subject sequence Escherichia coli str. substr. MG1655 (http://www.ncbi.nlm.nih.gov/nucleotide/556503834?report=genbank&log$=nuclalign&blast_r ank=1&RID=JUYB76FX014), and each of any matched chromosomal sequence is mapped against the bla sequence for counting the seed sequence from the canonical PAM (NGG) sequence. Secondary structures can be predicted by mFold. (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) to choose the sequence whose G is large as possible, preferably to be positive for crRNA spacer secondary structure. The following is the way to select the appropriate spacer sequences from the bla sequence. Table 1. Candidate anti-protospacer sequence.
    Seq Location anti-protospacer seq SEQ ID NO MG1655 pCas9 pBR322 PAM GC ΔG
    Off targ Off targ Off targ
    05 83-102(F) TAGATAACTACGATACGGGA 100 CGGGA None None GGG 0.48 -0.40
    05 83-102(F) TAGATAACTACGATACGGGA 100 5 GGG 0.48 +0.50
    22 442-461(F) GATCGTTGTCAGAAGTAAGT 101 AGTAAGT AGTAAGT None TGG 0.43 -0.80
    22 442-461(F) GATCGTTGTCAGAAGTAAGT 101 7 7 TGG 0.43 -0.50
    30 647-666(F) ACTTTAAAAGTGCTCATCAT 102 TCAT ATCAT None TGG 0.35 -1.30
    30 647-666(F) ACTTTAAAAGTGCTCATCAT 102 4 5 TGG 0.35 -1.30
    35 767-786 (F) TTTACTTTCACCAGCGTTTC 103 GCGTTTC TTTC None TGG 0.43 +1.30
    35 767-786 (F) TTTACTTTCACCAGCGTTTC 103 7 4 TGG 0.43 +2.20
    65 231-250 (RC) ATTAATAGACTGGATGGAGG 104 GATGGAGG GAGG GAGG CGG 0.48 +0.00
    65 231-250 (RC) ATTAATAGACTGGATGGAGG 104 8 4 4 CGG 0.48 +0.90
    66 234-253 (RC) ACAATTAATAGACTGGATGG 105 GATGG None None AGG 0.39 -0.30
    66 234-253 (RC) ACAATTAATAGACTGGATGG 105 5 AGG 0.39 +0.20
    67 237-256 (RC) GCAACAATTAATAGACTGGA 106 GACTGGA CTGGA CTGGA TGG 0.39 -0.20
    67 237-256 (RC) GCAACAATTAATAGACTGGA 106 7 5 5 TGG 0.39 -0.20
    68 241-260 (RC) CCCGGCAACAATTAATAGAC 107 AATAGAC AGAC None TGG 0.48 +1.80
    68 241-260 (RC) CCCGGCAACAATTAATAGAC 107 7 4 TGG 0.48 +2.60
    69 259-278 (RC) AACTACTTACTCTAGCTTCC 108 AGCTTCC GCTTCC None CGG 0.48 +0.60
    69 259-278 (RC) AACTACTTACTCTAGCTTCC 108 7 6 CGG 0.48 +1.60
    70 284-303 (RC) ACGTTGCGCAAACTATTAAC 109 TATTAAC None TAAC TGG 0.43 -1.90
    70 284-303 (RC) ACGTTGCGCAAACTATTAAC 109 7 4 TGG 0.43 -1.90
    73 375-394 (RC) TGTAACTCGCCTTGATCGTT 110 TCGTT CGTT CGTT GGG 0.52 -0.50
    73 375-394 (RC) TGTAACTCGCCTTGATCGTT 110 5 4 4 GGG 0.52 +0.10
    74 376-395 (RC) ATGTAACTCGCCTTGATCGT 111 TTGATCGT TCGT None TGG 0.48 -0.50
    74 376-395 (RC) ATGTAACTCGCCTTGATCGT 111 8 4 TGG 0.48 +0.10
    81 443-462(RC) AACTTACTTCTGACAACGAT 112 CAACGAT None None CGG 0.43 +0.40
    81 443-462(RC) AACTTACTTCTGACAACGAT 112 7 CGG 0.43 +0.40
    84 528-547(RC) AGTCACAGAAAAGCATCTTA 113 GCATCTTA None None CGG 0.43 -0.50
    84 528-547(RC) AGTCACAGAAAAGCATCTTA 113 8 CGG 0.43 +0.50
    90 638-657(RC) ACTTTTAAAGTTCTGCTATG 114 GCTATG None None TGG 0.35 -0.70
    90 638-657(RC) ACTTTTAAAGTTCTGCTATG 114 6 TGG 0.35 +0.10
  • All the target sequence from the bla gene was obtained using Guide RNA Target Design Tool (https://wwws.blueheronbio.com/external/tools/gRNASrc.jsp) from BlueHeron. There are 98 target candidate sequences returned. Bacterial off-target chromosomal short similar sequences are mapped against the bla gene followed by counting the seed sequence from the canonical PAM sequence. Choose the sequences whose seed sequence number is less than eight and whose Gibbs free energy is relatively large. The summary of the property of the selected target sequences is shown in Table 1. This table also shows nucleotide length of the seed sequence of the off-target sequences on pCas9 and pBR322.
    • Oligo cassette sequence for spacer sequence
  • The following four spacer sequences are crRNA generating cassettes targeting beta-lactamase on pBR322 in E. coli as a host strain, which meet the above two criteria. crRNA CR05 cleaves phosphodiester bond between 762nd base C and 763rd base C, CR30 cleaves phosphodiester bond between 198th base G and 199th base A, CR70 cleaves phosphodiester bonds between 575th base T and 576th base A and CR90 cleaves phosphodiester bonds between 221st base T and 222nd base A on the beta-lactamase gene.
  • Adaptors for single targets on the beta-lactamase gene.
    • CR05
      5'-AAACTAGATAACTACGATACGGGAg SEQ ID NO: 115 atctattgatgctatgccctcAAAA-5' SEQ ID NO: 116
    • CR30
      5'-AAACACTTTAAAAGTGCTCATCATg SEQ ID NO: 117 tgaaattttcacgagtagtacAAAA-5' SEQ ID NO: 118 DraI
    • CR70
      5'-AAACACGTTGCGCAAACTATTAACg SEQ ID NO: 119 tgcaacgcgtttgataattgcAAAA-5' SEQ ID NO: 120 AclI
    • CR90
      5'-AAACACTTTTAAAGTTCTGCTATGg SEQ ID NO: 121 tgaaaatttcaagacgataccAAAA-5' SEQ ID NO: 122 DraI
  • Adaptor for dual targets on the beta-lactamase gene.
    Internal direct repeat sequence is italicised and underlined.
    CR30+CR90 SEQ ID NO: 123
    Figure imgb0001
  • The 5' end of each oligo is phosphorylated and ready for cloning at BsaI sites.
    Sites of six base cutter restriction endonucleses are underlined, which are useful to screen the recombinants. We can also employ one of the cassette oligos as a primer to screen the recombinants by PCR together with another unique primer for the plasmid vector.
    • Appendix 1 pCas9 plasmid sequence (SEQ ID NO: 125)
  • Cas 9 gene, CRISPR expression locus and tracrRNA (all from S. pyogenes)
    Figure imgb0002
    Figure imgb0003
    Figure imgb0004
    Figure imgb0005
    Figure imgb0006
  • The reverse complement of tracrRNA is in lowercase, Cas9 coding sequence (SEQ ID NO: 126) is boxed and direct repeat sequence in CRISPR is in bold. Promoter sequences were predicted by neural network algorithm (http://www.fruitfly.org/seq_tools/promoter.html). The two unique sites, Sal I (GTCGAC) and Xba I (TCTAGA) highlighted in bold italicised black are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18. The pACYC184 backbone sequence is italicised.
  • The information of Appendix 1 above is alternatively presented as follows.
    • pCas9 plasmid sequence (SEQ ID NO: 125)
  • Cas 9 gene, CRISPR expression locus and tracrRNA (all from S. pyogenes)
    Figure imgb0007
    Figure imgb0008
    Figure imgb0009
  • Backbone vector pACYC184 sequence is italicised, sequence positions are numbered from G of EcoRI site underlined. The reverse complement of tracrRNA is in italicised bold located from 1844 to 1929, Cas9 initiation and termination codons are indicated by bold three letters, starting at nucleotide No. 2225 and ending at 6331 followed by leader sequence 6389-6483 indicated by italicised bold letters, first, second and third direct repeat sequences are underlined, between the first and second direct repeat in which spacer cloning region is located.
  • This spacer cloning region contains two inverted BsaI sites indicated by bold italicised letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3', respectively. Promoter sequences were predicted as above, indicated by lower case for forward promoter for Cas9 and leader sequence and italicised lower case for reverse promoter for tracrRNA, the putative transcription start site is indicated by bold uppercase. The two unique sites, Sal I (GTCGAC) and Xba I (TCTAGA) highlighted in bold italicised black are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18.
    • Appendix 2 Modification of spacer cloning site on pCas9
  • This system exploits the fact that expression of a minigene yields a certain oligopeptide that depletes the tRNA pool in the host cell, when induced for expression, which leads to the disruption of protein synthesis (Tenson T, Vega-Herrera J, Kloss P, Guarneros G, Mankin AS. J. Bacteriol. 1999; 181:1617-1622). Applying this concept to cloning the spacer sequence: when the spacer sequence is inserted this interrupts the minigene and the oligopeptide expression does not occur, thus tRNA is not depleted, thus only the bacteria harbouring a plasmid with insert can survive under conditions of induced expression; whilst bacteria harbouring the plasmid without insert cannot grow. The following (SEQ ID NO: 129) is the sequence structure. In order to construct this structure, bacteria should express the lac I repressor constitutively to switch off minigene transcription. Recombinant selection should be performed in the presence of IPTG to induce transcription.
    Figure imgb0010
  • BsaI recognition sites are underlined. lac operator 3 and 1 are bold italicised. Tac promoter is in bold letters, -35 and -10 region in the promoter sequence are underlined, Shine-Dalgarno sequence is boxed, first dipeptide is in bold M and R followed by three consecutive termination codons italicised TAA. Tryptophan terminator signal sequence is employed, indicated by italicised letters.
    • Appendix 3 Expected cleavage site on target sequence using CR90 spacer sequence First processing event
  • tracrRNA hybridises to the direct repeat region of pre-crRNA indicated by upper case letters using the sequence underlined. Bacterial RNase III cleaves the double-stranded RNA region at indicated position "I", first processing event.
    Figure imgb0011
    • Structure after 1st processing event
  • Figure imgb0012
  • The first processing event may also be depicted as follows: tracrRNAs indicated by lower case letters hybridise to the direct repeat region of pre-crRNA indicated by upper case bold black letters. Bacterial RNase III cleaves the double-stranded RNA region at indicated position with arrows, which is first processing event. Phosphodiester bond between 22nd and 23rd base in the first and second direct repeat are cleaved. Italicised spacer sequence CR90 is boxed.
    Figure imgb0013
    • Second processing event
  • The 2nd cleavage point (*) is around 20 nt away from the 3' end of the spacer sequence. "Note that the 2nd processing event occurs at a specific distance from the 1st cleavage within the repeats. Considering that spacer sequences are not identical among each other, it is thus likely that the 2nd processing event within the spacers is distance-dependent rather than sequence-dependent" (see Supplementary figure 2 legend in Nature. ).
  • Although the above-referenced article does not particularly specify the enzyme and/or mechanism involved in this 2nd processing event, it is most likely Cas9 is involved in this 2nd cleavage. Considering this cleavage position within the spacer sequence, the whole spacer sequence is not contributing to the target recognition, instead only 20 nt spacer sequence is utilised for hybridisation.
    Figure imgb0014
  • The second processing event may also be depicted as follows: The 2nd cleavage point indicated by arrows around 20 nt away from the 3' end of the spacer sequence in the above figure.
  • The above note quoted from Nature, and our comment, applies.
    The italicised spacer sequence CR90 is in box.
    Figure imgb0015
    • Target DNA cleavage
  • The part of the target beta-lactamase DNA sequence containing CR90 is shown. crRNA hybridises to its complementary sequence of the target region, Cas9 cleavage points are indicated by dot "." and the PAM sequence tgg is indicated in the box.
    Figure imgb0016
  • An alternative depiction of the target DNA cleavage is shown below. Here, the part of the target beta-lactamase DNA sequence containing CR90 proto-spacer sequence is shown in lower case bold black letters. crRNA hybridises to its complementary sequence of the proto-spacer strand, Cas9 cleavage points are indicated by dot arrows and the PAM sequence tgg is under lined.
    Figure imgb0017
    • Example 2
  • The following experiments describe some proof-of-concept experiments performed to demonstrate that the CRISPR-Cas9 system can be used to inactivate antibiotic resistance in bacteria. They describe the construction of a generally applicable DNA cassette, described in the Examples to deliver the CRISPR-Cas9 system plus a derivative carrying a spacer sequence targeted against an antibiotic resistance gene for delivery by naked DNA transformation and bacteriophage infection and also to demonstrate inhibition of the spread of antibiotic resistance by plasmid conjugation.
    • Construction of pNB100
  • pNB100 is a vector to express the CRISPR-Cas9 system in E.coli with the appropriate unique restriction site, Bsa I, to clone any desired spacer sequence between two direct repeats in the CRISPR locus. The backbone of the vector is derived from pACYC184 and the CRISPR-cas9 locus is inserted into Eco RV site of the vector. Three regions of the CRISPR-cas9 locus were amplified by PCR from the genomic DNA of Streptococcus pyogenes strain SF370, purchased from the ATCC, and assembled by Gibson assembly (Gibson DG, et.al. Nature Methods 2009; 6: 343-345) along with the pACYC184 vector in the reaction. The sequence of the final construct was verified by Sanger sequencing. The CRISPR-Cas9 activity was confirmed using a derivative of pNB100, pNB102, carrying a spacer sequence targeted against the beta lactamase genes of the bacterial transposons Tn3 and Tn1.
    • The amplified sequence of the three regions and amplicon image on the gel
  • The following sequences are the three regions amplified by PCR. Underlined sequences are template-specific primer sequences, bold letters are overlapping sequences used for Gibson assembly.
    1. 1. Fragment 1 (SEQ ID NO: 140), tracrRNA-cas9 : amplicon length = 4758 bp Forward primer is from 854170 to 854193 and reverse primer is from 858867 to 858848 on S. pyogenes SF370 genomic DNA.
      Figure imgb0018
      Figure imgb0019
    2. 2. Fragment 2 (SEQ ID NO: 141), Leader and first direct repeat: amplicon length = 276 bp Forward primer is from 860648 to 860671 and reverse primer is from 860862 to 860806 on S. pyogenes genomic DNA.
      Figure imgb0020
    3. 3. Fragment 3 (SEQ ID NO: 144), Second direct repeat: amplicon length = 452 bp Forward primer is from 861221 to 861276 and reverse primer is from861613 to 861594 on S. pyogenes genomic DNA. Decamer sequence from 861246-861255 GGTCTCCATT (SEQ ID NO: 142), which contains BsaI recognition sequence on the genomic DNA, was substituted with GGTCCCAAAA (SEQ ID NO: 143) to destroy BsaI recognition sequence and convert the 7th truncated direct repeat in the CRISPR array on the genome to the canonical 2nd direct repeat sequence in this vector.
      Figure imgb0021
  • PCR conditions to generate the three fragments were:
    Fragment 1 Fragment 2 Fragment 3
    5X Q5 Reaction Buffer 1 x 1 x 1 x
    10 mM dNTPs 200 µM 200 µM 200 µM
    10 µM Forward Primer 0.5 µM 0.5 µM 0.5 µM
    10 µM Reverse Primer 0.5 µM 0.5 µM 0.5 µM
    S. pyogenes DNA 50 - 100 ng/ul 1 ng/µl 1 ng/µl 1 ng/µl
    Q5 High-Fidelity DNA Polymerase 2 U/µL(NEB) 0.04 U/µl 0.02 U/µl 0.02 U/µl
    Thermocycling condition
    Initial Denaturation 98°C_60 sec 98°C_60 sec 98°C_60 sec
    35 Cycles 98°C_10 sec 98°C_10 sec 98°C_10 sec
    64°C_30 sec 62°C_30 sec 62°C_30 sec
    72°C_240 sec 72°C_30 sec 72°C_30 sec
    Final Extension 72°C_120sec 72°C_120 sec 72°C_120 sec
    Hold
    4 °C 4 °C 4 °C
  • Results showing the PCR amplicons are provided in Figure 11.
    • Assembly of pNB100 from three PCR amplicons, tracrRNA-cas9, leader and first direct repeat, second direct repeat; plus pACYC184 digested with EcoRV
  • We employed a Gibson assembly kit from NEB (E5510) and followed the protocol provided by the manufacturer to assemble the above three PCR amplicons along with pACYC184. The component of each fragment in the assembling reaction is shown in the following table.
    0.1 pmol/µL Fragment 1 0.2 pmol
    0.2 pmol/µL Fragment 2 0.2 pmol
    0.2 pmol/µL Fragment 3 0.2 pmol
    0.01 pmol/µL pACYC184 0.04 pmol
    Fragments 1 :: Fragment 2 : Fragment 3 : vector 5: 5 : 5 : 1
    Gibson Assembly Master Mix (2X) 1x
    Incubation 50°C for 1 hr
  • 2 µL of the assembly reaction was transformed to DH5α competent cells (purchased from New England Biolabs) followed by selection on chloramphenicol (35µg/mL) LB plates. The recombinants were screened by PCR using the three primer sets used for obtaining the initial three fragments. The plasmid templates giving three desired amplicons were isolated from the candidate clones and were subjected to sequence analysis.
    • The sequence of the final construct of pNB100 (SEQ ID NO: 145)
  • Figure imgb0022
    Figure imgb0023
    Figure imgb0024
  • The total number of nucleotides is 9578 bp. The backbone vector pACYC184 sequence is italicised, sequence positions are numbered from G of EcoRI site (GAATTC) underlined. tracrRNA is located at nucleotide No. from 1889 to 1974 indicated bold letters, Cas9 initiation and termination codons are indicated by bold three letters, starting at nucleotide No. 2270 and ending at 6376 followed by leader sequence 6462-6556 indicated by italicised bold letters, first and second direct repeat sequences are underlined, between which spacer cloning region (30 mer) is located. This spacer cloning region contains two inverted BsaI sites indicated by bold italicised letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3', respectively and one unique AatII (5'-GACGTC-3') site also indicated by bold italicised to reduce self-ligation in the event of incomplete BsaI digestion. Note the transition and transversion base changes G6573A, A6779T that were detected by Sanger sequencing and are shown in bold letters, respectively. However, these point mutations do not affect the CRISPR-Cas9 activity, which will be shown in the later section. The two unique sites, SalI (GTCGAC) and XbaI (TCTAGA) highlighted in bold italicised letters are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18.
  • A plasmid map of pNB100 is shown in Figure 12.
  • The desired spacer sequence can be cloned in the clockwise direction between BsaI sites. This vector contains the p15A origin at 1393-848 and cat (chloramphenicol resistant) gene at 219-9137. Cutting positions of each restriction enzyme, indicated in the parentheses, refer to the position of the 5' cutting sites on the top strand within the recognition sequence.
    • Construction of pNB102
  • pNB100 was digested with BsaI and AatII followed by purification using Agencourt ampure beads. The spacer sequence CR30 was employed from the discussion in B. Selection of spacer sequence from the target sequence in the Materials and Methods section above. The CR30 sequence is as follows :
    • 5'-AAACACTTTAAAAGTGCTCATCATg (SEQ ID NO: 117)
    • tgaaattttcacgagtagtacAAAA-5' (SEQ ID NO: 118)
  • This double-stranded DNA cassette is generated by denaturation at 95 C for 1 min and re-annealing at -1 degree every min to 30 C in the 1 x T4 ligase buffer (50 mM Tris-HCI(pH 7.5 at 25 C), 10 mM MgCl2, 1 mM ATP 10 mM DTT) plus 50 mM NaClfollowing the kinase reaction to add a phosphate moiety in the 5' terminus of each oligo. The annealed cassette contains 5' protruding four base compatible bases on both ends for the sites created on pNB102 by BsaI digestion. This CR30 cassette was ligated to pNB100 by T4 DNA ligase and transformed to DH5α competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the bottom sequence of the CR30 cassette as a reverse primer and a forward primer CF1: 5'-acgttgacgaatcttggagc, which anneals at 6209-6228 region on the recombinant plasmid to generate 409 bp PCR amplicon. PCR positive clones were sequenced to confirm the CR30 spacer sequence and this recombinant clone is designated as pNB102 and used for in vitro beta lactamase-gene disruption experiments. CR30 spacer anneals to the sense strand of beta lactamase-gene and cleaves the phosphodiester bonds between 188th and 189th nucleotide on the sense and antisense strand.
    • Construction of M13mp18::NB102
  • pNB102 was digested with unique restriction sites SalI and XbaI to generate two fragments 6044 bp and 3524 bp. The fragment length was calculated from the 5' end of the restricted sites in the top strand within the restriction recognition sites. The 6044 bp fragments containing CRISPR locus with CR30 spacer sequence in the CRISPR array was separated from the 3524 bp fragment and purified on the preparative 1 % agarose gel. M13 mp18 was digested with SalI- XbaI, followed by Agencourt ampure purification to remove the six bases SalI-XbaI fragment from the reaction. These two purified fragments were combined and ligated by T4 DNA ligase and transformed to DH5αF'Iq competent cells (purchased from New England Biolabs). Transformed cells were plated along with freshly grown DH5αF'Iq cells as a phage indicator and IPTG/X-gal solution as a blue-white colour indicator with molten top agar to LB plate. White plaques collected from the lawn were screened by PCR for the presence of the CR30 spacer sequence. The correct phage constructs obtained were purified by two times single plaque isolation. The entire sequence length of the final construct is 13288 bp. This spacer CR30 positive recombinant is designated as M13mp18::NB102 and was used for the bla-gene inactivation experiments mediated by M13 phage delivery.
    • Delivery of CRISPR-Cas9 constructs to bacteria
  • Having constructed the CRISPR-Cas9 plasmid pNB100 and the derivative plasmid pNB102 carrying a spacer insertion targeted against the beta-lactamase (bla) genes encoded by the bacterial transposable elements Tn1 and Tn3, we then sought to demonstrate, using three delivery methods, (i) plasmid conjugation, (ii) plasmid DNA transformation, (iii) bacteriophage infection, that bacterial cells carrying copies of the CRISPR-Cas9 construct with bla-spacer insertion would be unable to grow in the presence of the beta-lactam antibiotic ampicillin.
  • Constructs that are able to inactivate target genes, including antibiotic resistant genes, via the CRISPR-Cas system, and which are an aspect of the present invention are also referred to herein as "Nemesis symbiotics".
    • "Nemesis symbiotic activity" (NSA) assay by plasmid conjugation: a prophylaxis experiment
  • We demonstrate here that Nemesis symbiotics can prevent the spread of antibiotic resistance by inhibiting conjugal transfer of conjugative plasmids carrying antibiotic resistance genes from a donor cell to a recipient cell carrying the Nemesis symbiotics. To do this we mated a recipient cell carrying the Nemesis symbiotics with a donor cell carrying a conjugative plasmid, plus a multicopy mobilisable plasmid carrying the bla gene encoding ampicillin resistance. In a successful mating, the conjugative plasmid will transfer itself plus the mobilisable plasmid carrying ampicillin resistance to the recipient. Exconjugants may be selected for resistance to both chloramphenicol present on a non-mobilisable plasmid in the recipient and ampicillin received from the donor. Successful Nemesis symbiotic activity will reduce the efficiency of transfer of ampicillin resistance.
  • The recipient cell DH5α (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ-) was purchased from New England Biolabs and transformed with the plasmids pNB100 or pNB102 or pACYC184, where plasmids encode chloramphenicol resistance and both pNB100 and pNB102 carry CRISPR-Cas9 but only pNB102 carries the spacer sequence targeted against the beta-lactamase gene. The plasmid pACYC184 is the non-mobilisable parent plasmid used for the construction of pNB100 and pNB102 as described above.
  • The donor strain JA200 (F+ thr-1, leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl) also carrying plasmid pNT3 is described by Saka et al. ). The plasmid pNT3 is a mobilisable plasmid carrying the bla gene of Tn1.
  • A single colony of the donor JA200 pNT3 was picked from a Luria broth (LB) plate containing 100 µg/mL Ampicillin and grown shaking at 37°C overnight in 1mL LB medium with 100 µg/mL Ampicillin. A single colony each of the recipients, DH5α pNB100 and DH5α pNB102 was picked from a LB plate containing 35 µg/mL Chloramphenicol and grown shaking at 37°Covernight in 1mL LB with 35 µg/mL Chloramphenicol. To wash cells to remove antibiotics, 50µL of cells were added to 1mL LB in Eppendorf tubes and centrifuged 60 sec at 12500 rpm. Cells were resuspended in 50µL LB. To set up the mating JA200 pNT3 was spotted onto an LB plate, then 2µL of each DH5α carrying pNB100 and pNB102 were added to this spot. Separate 2µL spottings of donor and recipients were also performed (i.e. not mated). Plates were incubated at 37°C for 4 hours. Cells were removed resuspended in LB and 100µL plated on LB plates containing both 100 µg/mL Ampicillin and 35 µg/mL Chloramphenicol (LB ApCm). 100µL of 10,000 fold (10^-4) dilutions were also plated on LB plates and incubated at 37°C overnight. The resultant colonies were counted as shown in the Table below.
    Cells LB ApCm plates LB plates 10∧-4 dilution Nemesis symbiotic activity
    JA200 pNT3 x DH5α pNB100 Confluent Approx. 500 Negative
    JA200 pNT3 x DH5α pNB102 37 Approx. 500 Positive
    JA200 pNT3 x DH5α pACYC184 Confluent Approx. 500 Negative
    JA200 pNT3 0 Not done Not applicable
    DH5α pNB100 0 Not done Not applicable
    DH5α pNB102 0 Not done Not applicable
    DH5α pACYC184 0 Not done Not applicable
  • Photographs in Figure 13 show platings of the matings between: (A) JA200 pNT3 x DH5α pNB100 (as expected lacking Nemesis symbiotic activity); and (B) JA200 pNT3 x DH5α pNB102 (showing Nemesis symbiotic activity).
  • The 10^-4 dilution plated on LB plates, gave approximately 500 colonies a count of 5 x 10^7 cells per mL in the mated cell suspension and for the JA200 pNT3 x DH5α pNB102 only 3.7 x 10^ 2 cells/mL were able to grown on the LB Ap1 00Cm35 plates. Thus assuming half the cells are recipients/ex-conjugants then: 3.7 x 10^ 2 cells /ml divided by 2.5 x 10^7 gives a mating efficiency for the recipient carrying pNB102 of 1.2 x 5 x 10^ -5
  • The experiment demonstrated a significant reduction in CmRApR exconjugants in matings with DH5α carrying pNB102 versus pNB100 x JA200 pNT3.
  • In order to measure the relative mating efficiencies more accurately, after a liquid mating cells were plated on LB plates to titre all cells, LB Ap100 plates to titre donors plus exconjugants, LB Cm plates to titre recipients plus exconjugants and LB Ap100Cm35 to titre exconjugants only.
  • For the liquid mating overnight cultures of 10µL of JA200 pNT3 were mixed with 10µL of recipients DH5α pNB100 or DH5α pNB102 200µL of LB added and tubes incubated overnight at 37°C. Mating mixtures were diluted 10^-1, 10^-3, 10^-5 in LB and 50µL of dilutions plated on LB, LB Ap100Cm35, LB Ap100 and LB Cm35 plates and plates incubated overnight at 37°C. The table below summarises the cell titres obtained.
    Mated with JA200 pNT3 LB All cells LB Ap Cm Exconjugants LB Cm recipients and exconjugants LB Ap donors and exconjugants mating effic/donor mating effic/recipient
    pNB100 4.14 x 10 ^8 2.80 x 10 ^7 1.40 x 10 ^8 2.64 x 10 ^8 1.06 x 10^ -1 2.00 x 10^ -1
    pNB102 5.16 x 10 ^8 7.20 x 10 ^3 1.78 x 10 ^8 3.82 x 10 ^8 1.88 x 10^-5 4.04 x 10^ -5
  • The number of cells on LB Cm plus LB Ap plates should equal the number of cells on LB plates. For pNB100 1.40 x 10^8 (Cm plates) plus 2.64 x 10^8 (Ap plates) = 4.04 x 10^8 which agrees very well with 4.14 x 10^8 on LB plates. For pNB102 1.78 x 10^8 (Cm plates) plus 3.82 10^8 (Ap plates) = 5.6 x 10^8 which agrees very well with 5.16 x 10^8 on LB plates.
  • In conclusion, the data show that after overnight mating in liquid culture, there is a 5,000 fold reduction in mating efficiency per recipient comparing pNB102 with the spacer to pNB100 lacking the spacer.
    • NSA Assay by plasmid transformation
  • In this experiment, we demonstrate that introduction of Nemesis symbiotics to recipient cells by DNA transformation inactivates antibiotic resistance in the transformants.
  • In order to obtain a tester strain, DH5α competent cells purchased from New England Biolabs were transformed with pBR322 (carrying the bla gene derived from Tn3) and selected on LB Ap100 plates. Competent cells of the derived strain DH5α pBR322 were then prepared using the CaCl2 protocol 25 (1.116) as described by Sambrook and Russell in Molecular Cloning: A Laboratory Manual (3rd Edition, 2001) and subsequently transformed with plasmids pNB100, pNB102 and pACYC184 with selection for CmR. Transformant colonies were then picked onto LB Cm35 and LB Ap100 plates. Primary transformants were replica toothpicked onto both LB Cm35 and LB Ap100 plates and incubated overnight at 37°C.
  • The results, depicted in Figure 14, show that all colonies toothpicked from DH5α pBR322 transformed by pNB100 (lacking the bla gene target spacer sequence) remain resistant to ampicillin. In contrast all colonies toothpicked from DH5α pBR322 transformed by pNB102 have lost ampicillin resistance, so demonstrating Nemesis symbiotic activity.
  • The experiments above do not give a value for the fraction of primary transformants where NSA has inactivated the bla gene. To address this, single colonies from the primary transformants were picked into 1mL LB and diluted 10^-3 in LB. Then 100µL plated onto plates as follows, and results scored. The results showed that following transformation of DH5α pBR322 with pNB102 fewer than 10^-6 cells retain ApR. Nemesis symbiotic activity is very efficient.
    • NSA Assay by bacteriophage M13 infection
  • In this experiment, we demonstrate that introduction of Nemesis symbiotics to recipient cells by bacteriophage infection inactivate antibiotic resistance in the transformants. We chose the male-specific filamentous phage M13 as the delivery agent for the Nemesis symbiotic construct. An M13 derivative M13mp18::NB102 carrying and the Cas9 CRISPR plus bla gene target spacer region of pNB102 was used to deliver the Nemesis symbiotic by infection of an F+ strain, JA200, carrying ampicillin resistance on the plasmid pNT3. 0.2mL of a fresh culture of this strain was added to 3 mL of LB top agar (Luria broth with 0.7% agar) and poured onto an LB plate. Then 2µL of phage stocks of M13mp18::NB102 (10^8 pfu/mL) and as a control M13mp18 were spotted onto the lawn and the plate was incubated 8 hours at 37°C. Plaques were picked into 1.5 mL LB and grown shaking o/n at 37°C. A control strain DH5α lacking ampicillin resistance was also cultured overnight from a single colony picked into 1.5 mL LB.
    • Nitrocefin assay for beta lactamase activity was performed:
  • 1mL of the culture of cells was centrifuged for 60 sec at 12,500 rpm in microfuge then 2µL of stock nitrocefin (10mg/mL in DMSO) was added to 1mL of cell supernatant and absorbance of the degradation product of nitrocefin was measured at 482 nm in a spectrophotometer several time points after addition of nitrocefin. The Table below summarises the results.
    Strain 30 sec 60 sec 2 min 5 min
    JA200 pNT3 infected by M13mp8 0.5 0.64 0.73 0.79
    JA200 pNT3 infected by M13mp8::NB102 0.08 0.09 0.11 0.15
    DH5α 0.09 0.07 0.06 0.05
  • The experiments reported above provide the proof-of-concept that, in the model organism, Escherichia coli, DNA constructs carrying the Cas9 CRISPR region plus a spacer region with sequences directed against a target region of the beta-lactamase gene can inactivate ampicillin resistance when delivered by naked DNA transformation and bacteriophage infection as well as prevent transfer of ampicillin resistance by plasmid conjugation. It is apparent that Nemesis symbiotics of the invention can be applied to pathogenic bacteria and for other antibiotic resistance genes.
    • Example 3
  • The aim of Example 3 is to extend the proof-of-concept for resurrection of antibiotic efficacy by introduction of a CRlSPR/Cas9 construct in a pathogenic bacterial strain of Klebsiella pneumoniae. Resistance to a newer class of beta-lactam antibiotics, the Carbapenems has emerged in Enterobacteriaceae by the acquisition of new bla genes that encode beta-lactamases able to degrade even the Carbpenems. Klebsiella pneumoniae strains with resistance to Carbapenems, KPC are important causes of morbidity and mortality among hospital-acquired and long-term care-associated infections. Noteworthy is the very high mortality (around 30-70%) among patients with bacteraemia or pulmonary infections.
  • Example 3 shows that a Klebsiella pneumoniae carrying a beta-lactamase (bla) gene conferring resistance to the beta-lactam antibiotic, ampicillin become sensitive to ampicillin following introduction of a modified CRISPR/Cas9 construct targeted against the bla gene. The same CRISPR/Cas9/anti-bla construct used in Example 1 or Example 2 is used and is delivered to Klebsiella pneumoniae by conjugation with a donor strain carrying a conjugative plasmid with CRlSPR/Cas9/anti-bla. Ex-conjugant recipient Klebsiella pneumoniae carrying the plasmid with CRlSPR/Cas9/anti-bla. construct is selected for on appropriate media with counter-selection against the donor cell and Klebsiella pneumoniae cells that failed to receive the plasmid.
  • As for Example 1 and Example 2, efficacy of the CRISPR/Cas9/anti-bla contstruct is evaluated by comparison to a negative control of a Klebsiella pneumoniae ex-conjugant strain that received a plasmid with CRISPR/Cas9 (i.e. lacking the anti-bla region). As in Example 1 and Example 2, the beta-lactamase activity can be detected by nitrocefin to count white versus red colonies. Also as in Example 1 and Example 2, alternatively beta lactamase activity is seen directly challenging the bacteria on the LB plate with/without ampicillin and measuring the fraction of the ampicillin sensitive colonies versus ampicillin resistant ones.
  • The identical experiment to that described above is also evaluated using instead a CRlSPR/Cas9/anti-blaCb, where the CRISPR/Cas9/anti-blaCb construct carries a region, anti-blaCb, targeted against a bla gene encoding a beta-lactamase able to break down carbapenems and present in that particular Klebsiella pneumoniae strain.
    • Example 4
  • Example 4 provides delivery routes for the therapeutic constructs. These routes all apply to veterinary as well as human applications to be delivered orally, topically, probiotically and for use in surgical irrigation fluids and wound dressings.
  • Orally: Phage containing assassin construct as the active ingredient may be administered orally as a stabilised therapeutic preparation: either - administered before antibiotic therapy or administered in the form of an adjuvant complexed to an antibiotic. Conjugative plasmids containing assassin construct as the active ingredient may be administered as a culture of commensal bacteria carrying these plasmids in order to transmit these plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens.
  • Topically: Phage containing assassin construct as the active ingredient may be administered topically as a stabilised medication (ointment, spray, powder etc): either - administered before antibiotic topical medication; or administered in the form of a complex with an antibiotic medication. Topical application of conjugative plasmids containing assassin construct as the active ingredient may be via a stabilised culture of commensal bacteria carrying these plasmids in order to transmit the plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens.
  • Probiotically: Phage containing assassin construct as the active ingredient may be administered probiotically as a stabilised culture of commensal bacteria (e.g. Lactobacillus spp) carrying these plasmids in order to transmit the plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens. One example of this may be the probiotic, prophylactic administration of such a preparation to patients in settings with a high risk of infection with antibiotic resistant bacterial pathogens such as hospitals, care homes, schools, transplantation centres etc. Another example may be the administration of such a preparation to livestock via animal feeds, thereby limiting the rise and horizontal transmission of antibiotic resistant bacteria. The use of the present invention in livestock thus represents one means for (indirect) prophylactic treatment of antibiotic resistant bacteria in humans.
  • Surgical irrigation fluids: Phage containing assassin construct as the active ingredient may be added to surgical irrigation fluids and also sprays for disinfecting fomites. Stabilised commensal bacterial cultures containing conjugative plasmids incorporating assassin constructs may be added as coatings to surgical wipes and to wound dressings.
    • Example 5
  • In this example, a construct is designed to include multiple RNA guide molecules where each RNA guide molecule is transcribed by its own promoter.
  • Figure 16 exemplifies a set of spacer sequences that we have identified encoding 20 guide RNA molecules targeted against 117 different bla genes identified in the NCBI ARDB database for Klebsiella pneumoniae. The beta lactamase gene type, spacer sequence and antibiotic resistance profile in Klebsiella pneumoniae obtained from NCBI ARDB database are shown.
  • Beta lactamase gene sequences were collected from the ARDB database with the keyword Klebsiella pneumoniae. Redundant sequences were removed and unique sequences used for multiple sequence alignment using web program Clustal Omega. One canonical sequence was chosen from each cluster and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder collected. The spacer sequence was chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch. Each of the example spacer sequences shown in the 4th column has the capability to disrupt the genes in the third column.
  • Beta lactamase genes used in this analysis are: SHV-a = 1, 2, 2a, 5, 5a, 11, 12, 14, 26, 27, 28, 31, 33, 38, 43, 44, 48, 55, 56, 60, 61, 62, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,85, 89, 92, 98, 99, 101, 103, 106, 107, 108, 109, CTXM-b = 1, 3, 10, 12, 15, 22, 32, 54, 60, 62, 68, CTXM-c = 13, 14, 16, 18, 19, 24, 26, CTXM-d = 2, 35, 59, CTXM-e = 26, 63, TEM-f = 1, 1b, 3, ESBL, 139, KPC-g = 1, 2, 3, 4, OKP-h = A11, A12, A16, A17, B13, B-SB613, 6, LEN-i = 2, 17, 18, 19, 20, 21, 22, 24,GES-j = 1, 3, 4, 5, VIM-a = 1, 2, 4, 12, 19, IMP-b = 4, 8, CMY-a = 2, 4, 25, 31, 36, LAT-b = 1, 2, CMY-c = 1, 8b, 10, 19, FOX-d = 1, 5, 7, OXA-a = 1, 30, 47, OXA-2, OXA-9, OXA-b = 10, 17.
  • Beta lactam antibiotics were classified into four classes, penams, cephems, carbapenem and monobactam. One antibiotic name is listed in Figure 16 as an example under each class. The beta lactamase, which can open the beta lactam ring is indicated by R. For example, carbapenem is inactivated by KPC. To re-sensitise bacteria to carbapenem, the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG (SEQ ID NO: 45) should be employed into the spacer array and inactivate KPC genes. Note that the spacer sequence for CMY-a can be employed to LAT-b cleavage.
  • Similarly, Figure 17 exemplifies a set of spacer sequences encoding 17 guide RNA molecules targeted against 154 different bla genes identified in the CARD database for Klebsiella pneumoniae. Beta lactamase gene type, spacer sequence and antibiotic resistance profile in Klebsiella pneumoniae obtained from the IIDR CARD database are shown.
  • Beta lactamase gene sequences were collected by filtering all the collected beta lactamase genes with the keyword Klebsiella pneumoniae and subjected to multiple sequence alignment using web program Clustal Omega. One canonical sequence from each cluster was chosen and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder collected. The spacer sequence was chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch. The each of the example spacer sequences shown in the 4th raw has the capability to disrupt the genes in the third column.
  • Beta lactamase genes used in this analysis are: SHV-a = 1a, 5, 2a,11, 18, 20, 21, 22, 23, 28, 31, 32, 52, 55, 98, 99, 100, 106, 107, 110, 111, 121, 134, 136, 137, 140, 143, 144, 147, 148, 149,150, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162,163, 164, 165, 168, 172, 173, 178,179, CTXM-b = 10, 12, 15, 52, 54, 62, 71, 80, CTXM-c = 19, 81, 99, 147, TEM-f = 1, 162, 183, 192, 197, 198, 209, KPC-g = 3, 4, 6, 7, 8, 11, 12, 14, 15, 16, 17, OKP-h = 5, 6, A1, A2, A4, A5, A6, A7, A8, A9 A10, A11, A12, A13, A14, A15, A16, B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13, B17, B18, B19, B20, LEN-i = 5, 6, 7, 8, 9, 10, 11, 12, 13, 18, 19, 20, 21, 22, 23, 24, GES-j = 1, 3, 4, VIM-a = 4, 26, 27, 28, 33, 34, IMP-b = 32, 38, NDM-c= 1, 9, 10, ACT-3, CMY-a = 25, 31, 56, 87, FOX-d = 8, 9, OXA-a = 1, 30, 47, OXA-1, OXA-a = 181, 204, 247, OXA-9.
  • Beta lactam antibiotics are classified into four classes, penams, cephems, carbapenem and monobactam. In Figure 17, one antibiotic name is listed as an example under each class. The beta lactamase, which can open the beta lactam ring is indicated by R. For example, carbapenem is inactivated by KPC. To re-sensitize bacteria to carbapenem, the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG (SEQ ID NO: 45) should be employed into the spacer array and inactivate KPC genes.
    • Example 6
  • In this example, a modified Cas DNA-binding polypeptide is created that deletes and reseals rather than leaving a DSB.
  • As described above, DSBs caused by Cas DNA-binding polypeptide such as CRISPR-Cas9 can be lethal to the replicon targeted and can result in cell death rather than, for example, re-sensitisation to antibiotics, if an antibiotic resistance gene in the replicon is targeted for inactivation. Immediate cell death rather than such re-sensitisation to antibiotics for subsequent killing by antibiotic may increase selection pressure against delivery of the targeting constructs. Instead of DSBs, we may modify the Cas9 gene to allow the resealing of the targeted sequence after gene inactivation by introducing a deletion or inversion.
  • Tsai et al. (2014) have constructed a fusion between a catalytically inactive Cas9 (dCas9) protein to the wild-type nuclease domain of the restriction endonuclease Fok1 to increase the specificity of cleavage in eukaryotic cells. Proudfoot et al. (2011) have similarly fused zinc-finger DNA recognition domains to the catalytic domains of recombinases to programme site-specific recombination at designated DNA sequences.
  • We here add appropriate recombinase domains to dCas9 to catalyse a recombination reaction rather than a DSB at a CRISPR-mediated RNA-guided desired position on the target genes.
  • The Cas9 resolvase fusion, called Cas9R in Figure 18, is directed to the desired sites determined by the Cas9 domain:guide RNA spacer sequences; and the fused resolvase is positioned at the recombination site. To generate a deletion between two recombination sites: A and B, resolvases must dimerise at each recombination site. Thus two Cas9Rs need to be positioned in close proximity at each recombination site A1A2 and B1B2 as designated by the sequences encoded by the spacers S1-S4. The correct orientations of A1 relative to A2 and B1 relative to B2 will need to be determined experimentally.
  • The orientation of the gRNA as shown Fig. 19 is already proved in the case of dCas9 fused FokI (Tsai el al., 2014) - i.e. PAM sequences of each protospacer sequence in the recombination sites are directly positioned at the outer boundaries of the full-length recombination site. Thus, we employ the same configuration of the annealed gRNA polarity.
  • Figures 19 and 20 show a schematic process as to how this Cas9R fusion resolvase resolves the synapse and parental replicon DNA is recircularised.
    • Example 7
  • The following experiments describe some proof-of-concept experiments performed to demonstrate that the CRISPR-Cas9 system can be used in a single construct to inactivate a large number of different beta-lactamase genes that may be found amongst microbial pathogens as well as amongst the non-pathogenic members of the microbiome.
  • In these exemplifications, plasmids are constructed that carry the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against up to eight of the following beta-lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA.
  • For each of these eight families of beta-lactamase genes, a single spacer is designed that will target a number of gene members of that family. These are: SHV-a = 1, 1a, 2, 2a, 5, 5a, 11, 12, 14, 18, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 38, 43, 44, 48, 52, 55, 56, 60, 61, 62, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 85, 89, 92, 98, 99, 100, 101, 103, 106, 107, 108, 109, 110, 111, 121, 136, 134, 137, 140, 143, 144, 147, 148, 149, 150, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162, 163, 164, 165, 168, 172, 173, 178, 179; CTXM-b= 1, 3, 10, 12, 15, 19, 22, 32, 52, 54, 59, 60, 62, 68, 71, 80, 81, 99, 141, 147; TEM-c = 1, 1B, 3, 139, 162, 183, 192, 197, 198, 209; KPC-d = 1, 2, 3, 4, 6, 7, 8, 11, 12, 14, 15, 16, 17 ; VIM-e = 1, 2, 4, 19, 26, 27, 33, 34; IMP-f = 4, 8, 32, 38; and NDM-g = 1, 9, 10.
  • Figure 21 shows the eight spacer sequences that were designed to target the eight beta-lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48. The primer sequences used in the construction of the plasmids are listed in a table in Figure 22.
  • In one exemplification, a plasmid derivative of pNB100 (Figure 12), pNB104A (Figure 23), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against four beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: NDM, IMP, VIM and KPC.
  • In another exemplification, a plasmid derivative of pNB100, pNB104B (Figure 24), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against four beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: OXA-48, SHV, TEM and CTX-M.
  • In another exemplification, a plasmid derivative of pNB100, pNB108 (Figure 25), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against eight beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48.
  • In another exemplification, a plasmid derivative of pNB100 is constructed where spacer sequences can be expressed from a choice of two different promotors. This plasmid, pNB200 (Figure 26), carries a different unique restriction site downstream of each promotor to clone any desired spacer sequences flanked by their own direct repeats between two direct repeats in the CRISPR locus. Derivatives of pNB200 are described that carry the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against eight beta-lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48. In pNB202 (Figure 27), the second promotor is used to express spacers flanked by direct repeats to target the four beta-lactamase families of resistance genes: OXA-48, SHV, TEM and CTX-M. And in pNB203 (Figure 28), which is derived from pNB202, both promotors are used: the first to express spacers flanked by direct repeats to target four beta-lactamase families of resistance genes: NDM, IMP, VIM and KPC and the second promotor to express spacers flanked by direct repeats to target four beta-lactamase families of resistance genes: OXA-48, SHV, TEM and CTX-M.
    • Construction of pNB104A
  • The tetramer spacer concatemer a+b+c+d shown in Figure 29A was digested with BsaI, whose restriction site is located in A1 and A2, and ligated to BsaI spacer cloning sites on pNB100. The structure of the single promoter and spacer region (6221-7001) on pNB104A is shown in Figure 23. The four-space concatemer contains spacer sequences targeting NDM, IMP, VIM and KPC from the proximal to the distal end of the single promoter.
    • Construction of pNB104B
  • The tetramer spacer sequence concatemer e+f+g+h shown in Figure 29A was digested with SapI, whose restriction site is located in B1 and B2, and ligated to SapI spacer cloning sites on pNB200. While screening pNB202 screening this construct was found and confirmed by sequencing. A deletion event between the direct repeats adjacent to leader sequences had occurred and restored the direct repeat sequence to give pNB104B. The single promoter regions (6221-6987) on pNB104B is shown in Figure 24. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located under the single promoter.
    • Construction of pNB108
  • The concatenated spacer array sequences A and B were amplified from the subcloned vector pCR Blunt II-TOPO_SpacerA and pCR Blunt ll-TOPO_SpacerB with the primer set NB026 and NB029, NB030 and NB033, respectively. At the 3' end of amplicon of spacer A and 5' end of amplicon of spacer B are 20 bases of overlapped sequence from KPC spacer sequence. These two amplicons were gel purified and used for PCR-based pairwise cycle extension reaction in the absence of the primer. The extended material was re-amplified with primer set NB037 (5'-GGGCTGGCAAGCCACGTTTGGTG-3'; SEQ ID NO. 150) and NB038 (5'-CCGGGAGCTGCATGTGTCAGAGG-3'; SEQ ID NO. 151) to generate the full 8 spacer array concatemer. This eight-spacer concatemer was cloned into pCR Blunt II-TOPO vector and sequence was confirmed.
  • BsaI digestion of this pCR Blunt ll-TOPO subclone removes the full 8 spacer array concatemer as a subclone from the pCR Blunt ll-TOPO vector, which contains 5' protruding four base compatible bases on both ends for the sites created on pNB100 by BsaI digestion. Then pNB100 was digested with BsaI followed by agarose gel purification. The eight-spacer concatemer cassette, released from the pCR Blunt ll-TOPO was ligated into pNB100 by T4 DNA ligase and transformed to DH5α competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plates and were screened by PCR with the reverse primer NB021: 5'- GGTGACTGATGGCCACGT (SEQ ID NO: 149) and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC (SEQ ID NO: 148), which anneal at 6368-6385 region and 7203-7225 region, respectively on the recombinant plasmid to generate 858 bp PCR amplicon. PCR-positive clones were sequenced to confirm the eight-spacer concatemer sequence and this recombinant clone is designated as pNB108 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes. A plasmid map of pNB108 is shown in Figure 25.
    • Construction of pNB200
  • The plasmid pNB200 contains two identical promotors for gRNA expression and carries a different unique restriction site BsaI and Sapl downstream of each promotor to clone any desired spacer sequences flanked by their own direct repeats between two direct repeats in the CRISPR locus. The desired structure of the CRISPR array locus in pNB200 consists two sets of cassettes harbouring promotor-leader-direct repeat-spacer cloning region-direct repeat-tail tandemly. The forward primer NB018 anneals at the 5' end of the leader sequence to the promotor in pNB100 and introduces the first spacer cloning region, second direct repeat and tail sequence. The reverse primer NB019 anneals at the 3' end of leader sequence in pNB100 and introduces a third direct repeat, second spacer cloning region. This amplicon is cloned between BsaI sites on pNB100 to give pNB200.
    • The amplified sequence (SEQ ID NO: 146) with primer NB018 and NB019 from pNB100 as template
  • Figure imgb0025
  • The forward primer NB018 and the reverse primer NB019 are underlined and the initial annealing sites, for the first PCR cycle, are indicated by bold letters and italicised bold letters, respectively. This amplicon was digested with BbvI (5'-GCAGCN8/N12) to create four bases compatible protruding 5' ends to BsaI digested pNB100. BbvI recognition sites are depicted by lower bold cases.
  • The PCR conditions to generate the dual promotor-leader cassette were:
    Component
    Nuclease-Free water 41.25 µL
    10 X PCR Buffer 5 µL
    10 mM dNTPs 1 µL
    10 µM Forward Primer NB018 1 µL
    10 µM Reverse Primer NB019 1 µL
    pNB100 0.5 µL
    QIAGEN Hot Start Taq 0.25 µL
    • Cycle conditions
  • STEP TEMP TIME
    Initial Denat. 95CC 15 min
    35 Cycles 94CC 30 sec
    55 °C 30 sec
    72 °C 30 sec
    Final Extension 72°C 10 min
    • The sequence of the modified CRISPR array in pNB200 (SEQ ID NO: 147)
  • The total number of nucleotides of pNB200 is 9919 bp. The sequence in the modified region is shown below, which replaces the small BsaI fragment in pNB100. The first and the second promotor sequences are underlined. Leader sequences are in bold case. Direct repeats are italicised and the spacer cloning region between direct repeats are indicated by italicised and underlined.
  • The first spacer array cloning region contains two inverted BsaI sites indicated by bold italicised underlined letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3' on the vector, respectively and one unique AatII (5'-GACGTC-3') site also indicated by bold italicised underlined lettes to reduce self-ligation in the event of incomplete BsaI digestion.
  • The second spacer cloning region contains two inverted Sapl sites indicated by bold italicised underlined letters 5'-GAAGAGC-3' and 5'-GCTCTTC-3' for creating 5' three bases protruding spacer cloning sites 5'-GTT-3' and 5'-TTT-3' on the vector, respectively.
    Figure imgb0026
  • A plasmid map of pNB200 is shown in Figure 26. The desired spacer array sequences can be cloned in the clockwise direction between two BsaI sites and two SapI sites independently, for each promotor respectively.
    • Construction of pNB202
  • The plasmid pNB201 contains the dual promotors of pNB200 from which it is derived but a spacer array sequence targeted to OXA-48, SHV, TEM and CTX-M beta lactamase gene families are expressed from the second promotor.
  • To construct pNB202, pNB200 was digested with BsaI followed by agarose gel purification. The concatenated spacer array sequence B was generated by PCR-mediated pair-wise oligo nucleotide concatenation. This four-spacer concatemer array B was cloned to the pCR Blunt ll-TOPO vector, purchased from Life Technologies, and the sequence was confirmed. Sapl cuts out spacer array sequence B as a subclone from the TOPO vector and yields 5' protruding three base compatible bases on both ends for the sites created on pNB200 by Sapl digestion. This four-spacer concatemer B cassette was ligated to pNB200 by T4 DNA ligase and transformed to DH5α competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the reverse primer NB021: 5'- GGTGACTGATGGCCACGT (SEQ ID NO: 149) and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC (SEQ ID NO: 148), which anneal at 6368-6385 region and 7307-7329 region, respectively on the recombinant plasmid to generate 962 bp PCR amplicon. PCR positive clones were sequenced to confirm the four-spacer concatemer B sequence and this recombinant clone is designated as pNB202 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes. A plasmid map of pNB202 is shown in Figure 27.
    • Construction of pNB203
  • The plasmid pNB203 contains the dual promotors of pNB200 but each promotor expresses four spacers. The first promotor expresses spacer sequence targeted to NDM, IMP, VIM and KPC, the second promotor expresses OXA, SHV, TEM and CTX-M beta lactamase genes. The plasmid pNB202 was digested with BsaI followed by agarose gel purification. The concatenated spacer array sequence A was cut out from pCR Blunt ll-TOPO vector harbouring spacer concatemer A with BsaI. BsaI cuts out spacer region, which contains 5' protruding four base compatible bases on both ends for the sites created on pNB202 by BsaI digestion. This four-spacer concatemer A cassette was ligated to pNB202 by T4 DNA ligase and transformed to DH5α competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the reverse primer NB021: 5'- GGTGACTGATGGCCACGT and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC , which anneal at 6368-6385 region and 7479-7501 region, respectively on the recombinant plasmid to generate 1134 bp PCR amplicon. PCR positive clones were sequenced to confirm the four-spacer concatemer A and B sequence and this recombinant clone is designated as pNB203 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes. A plasmid map of pNB203 is shown in Figure 28.
    • Schematic representation of the structure of concatenated spacer arrays
  • Spacer sequences were determined to maximise the coverage of the target beta-lactamase gene family. Each unit oligo contains the direct repeat flanking the appropriate spacer sequence at each end. Concatenation reactions are performed between pairwise oligos, i.e. the nearest neighbour unit oligos are concatenated first to generate two unit length oligo, then two unit length oligos are concatenated to generate four unit length of oligo etc.
  • The schematic structure of tetramer and octamer spacer structures are shown in Figure 29. S: spacer, R: direct repeat, A and B contain BsaI site to create ligation compatible sites for cloning into pNB100 and downstream of the first promotor of pNB200. C and D contain a SapI site to create ligation a compatible site for cloning downstream of the second promotor of pNB200. In this example, we employed spacer sequence S1 targeting NDM, S2 targeting IMP, S3 targeting VIM, S4 targeting KPC, S5 targeting OXA, S6 targeting SHV, S7 targeting TEM and S8 targeting CTX-M.
    • Spacer concatenation reaction
  • Each oligo has overlapped sequence in the 3' and 5' end to anneal to the nearest neighbour oligo except the first and the last oligo. The first and the last oligo have the overlapping sequence to the second and the penultimate oligo in the 5' end only. In order to concatenate four spacers, four oligos are synthesised. In other words, oligo No.1 consists spacer 1 and 2 in the 5' and 3' ends. Oligo No.2 contains reverse complement of spacer 2 and 3 in the 3' and 5' ends. Oligo No.3 contains spacer 3 and 4 in the 5' and 3' ends. Oligo No.4 contains reverse complement of spacer 4 in the 3' end. Thus the oligo No.2 can link oligo No.1 and oligo No.3, oligo 4 anneals to 3' end of oligo No.3. Oligo No.1 and oligo No. 2, oligo No. 3 and oligo No. 4 are concatenated in a separate tube using the following PCR reaction conditions.
    Component A1 A2 B1 B2 x 4
    Nuclease-Free water 33.5 µL 33.5 µL 33.5 µL 33.5 µL 134
    5X Q5 Reaction Buffer 10 µL 10 µL 10 µL 10 µL 40
    10 mM dNTPs 1 µL 1 µL 1 µL 1 µL 4
    Q5 High-Fidelity DNA Polymerase 0.5 µL 0.5 µL 0.5 µL 0.5 µL 2
    10 µM Forward Primer NB026 2.5 µL
    10 µM Reverse Primer NB027 2.5 µL
    10 µM Forward Primer NB028 2.5 µL
    10 µM Reverse Primer NB034 2.5 µL
    10 µM Forward Primer NB035 2.5 µL
    10 µM Reverse Primer NB031 2.5 µL
    10 µM Forward Primer NB032 2.5 µL
    10 µM Reverse Primer NB036 2.5 µL
    • Cycle conditions
  • STEP TEMP TIME
    Initial Denat. 98°C 60 sec
    35 cycles 98°C 10 sec
    55°C 10 sec
    72°C 20 sec
    Final Extension 72°C 2 minutes
    Hold 4°C
  • In this example, NB026 and NB027, NB028 and NB034, NB035 and NB031, NB032 and NB036 are concatenated. Each concatenated product A1, A2, B1 and B2 was gel purified and set up the second concatenation reaction using the purified A1 and A2, B1 and B2 dimer product in the following PCR condition.
    Component A B x 2
    Nuclease-Free water 35.75 µL 35.75 µL 71.5
    10 X PCR Buffer 5 µL 5 µL 10
    10 mM dNTPs 1 µL 1 µL 2
    QIAGEN Hot Start Taq 0.25 µL 0.25 µL 0.5
    Gel extracted A1 4 µL
    Gel extracted A2 4 µL
    Gel extracted B1 4 µL
    Gel extracted B2 4 µL
    • Cycle conditions
  • STEP TEMP TIME
    Initial Denat. 95°C 15 min
    35 Cycles 94°C 30 sec
    A: 55 °C 30 sec
    72°C 30 sec
    Final Extension 72°C 10 min
  • These extention products were amplified by NB037 and NB038 with Q5 DNA polymerase. The final amplicons were cloned to pCR Blunt II TOPO vector and the concatemer sequences were confirmed.
  • In case of eight spacer concatenation, spacer concatemer A and spacer concatemer B on pCR Blunt II TOPO vector were amplified with primer pairs NB026 and NB029, NB030 and NB033, respectively and amplicons were gel purified. Purified spacer A and B were utilised as a long primer in the following cycle extension reaction.
    Component A
    Nuclease-Free water 35.75 µL
    10 X PCR Buffer 5 µL
    10 mM dNTPs 1 µL
    QIAGEN Hot Start Taq 0.25 µL
    Gel extracted A 4 µL
    Gel extracted B 4 µL
    • Cycle conditions
  • STEP TEMP TIME
    Initial Denat. 95 °C 15 min
    25 Cycles 94 °C 30 sec
    55 °C 30 sec
    72 °C 30 sec
    Final Extension 72 °C 10 min
    Hold
    4 °C o/n
  • These extention products were amplified by NB037 and NB038 with Q5 DNA polymerase. The final amplicons were cloned into pCR Blunt II TOPO vector and the concatemer sequences were confirmed.
    • Delivery of CRISPR-Cas9 constructs to bacteria
  • Constructs that are able to inactivate target genes, including antibiotic resistant genes, via the CRISPR-Cas system, and which are an aspect of the present invention are also referred to herein as "Nemesis symbiotics". The CRISPR-Cas9 plasmid derivatives, pNB104A, pNB104B, pNB202 and pNB203 all carry spacer insertions targeted against the selected families of beta-lactamase (bla) genes described, and so provide exemplars to demonstrate that a single plasmid construct possesses Nemesis symbiotic activity (NSA) and is therefore able to inactivate representative genes from all 8 different families of beta-lactam antibiotics. The plasmid pNB104A (see Fig. 23) is tested for its ability to inactivate representative genes members of the NDM, IMP, VIM and KPC families; pNB104B (see Fig. 24) and pNB202 (ss Fig. 27) are tested for their ability to inactivate representative genes members of the OXA, SHV, TEM and CTX-M families; and pNB108 (see Fig. 25) and pNB203 (see Fig. 28) are tested for their ability to inactivate representative genes members of the SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA families.
    • Nemesis symbiotic activity (NSA) assay by plasmid transformation
  • The NSA assay described in Example 2 showed that DNA transformation of an E. coli strain, DH5α also carrying the TEM-3 beta lactamase gene on the plasmid pBR322, with plasmid pNB102 converts the transformant to ampiciilin sensitivity (ApS). Here, the plasmid pNB102 encodes resistance to chloramphenicol and the DH5α (pBR322) transformants now carrying pNB102 were selected on LB Cm plates and then screened for ApS (see Fig. 14). Here, the plasmid pNB102, in expressing the CRISPR/Cas9 system with the spacer sequence encoding the gRNA targeting the TEM-3 gene, inactivated the TEM-3 gene. In contrast in a negative control experiment, when DH5α (pBR322) was transformed with the parental plasmid pNB100 carrying the expressing the CRISPR/Cas9 system but lacking the gRNA targeting the TEM-3 gene, no conversion to ApS occurred.
  • For exemplification purposes an equivalent experiment is described where plasmid derivatives of pBR322 are constructed where the TEM-3 is replaced by representative genes from the other 7 different families of beta-lactam antibiotics: SHV, CTX-M, KPC, VIM, IMP, NDM and OXA. Such genes are obtained from suitable bacterial strains carrying such genes. This allows a direct comparison to the proof of concept experiments described in Example 2 in isogenic genetic backgrounds.
  • A set of E. coli and K. pneumoniae strains carrying representative genes from these seven different families of beta-lactam antibiotics were purchased from Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, UK. These are: NCTC13368, a K. pneumoniae strain carrying the SHV-18 gene; NCTC13353 an E. coli strain carrying the CTX-M-15 gene; NCTC13438 a K. pneumoniae strain carrying the KPC-3 gene; NCTC13440 a K. pneumoniae strain carrying the VIM-1 gene; NCTC13476 an E. coli strain carrying an uncharacterised IMP gene; NCTC13443 a K. pneumoniae strain carrying the NDM-1 gene and NCTC13442 a K. pneumoniae strain carrying the OXA-48 gene. All seven genes encode beta lactamases that are also able to degrade and inactivate the penam class of antibiotics (see Fig. 16, 17). All strains were tested and, as expected, found to be resistant to the penam class antibiotic, ampicillin.
  • Beta lactamase coding sequences are amplified from the cell with appropriate forward and reverse primer set shown below:
    Strain NCTC No. Resistance gene Forward primer 5' to 3' Reverse primer 5' to 3'
    NBKp001 13443 NDM-1
    Figure imgb0027
    Figure imgb0028
    NBKp002 13442 OXA-48
    Figure imgb0029
    Figure imgb0030
    NBKp003 13368 SHV-18
    Figure imgb0031
    Figure imgb0032
    NBKp004 13440 VIM-1
    Figure imgb0033
    Figure imgb0034
    NBKp005 13438 KPC-3
    Figure imgb0035
    Figure imgb0036
    NBEc018 13476 IMP-4
    Figure imgb0037
    Figure imgb0038
    NBEc019 13353 CTX-M-15
    Figure imgb0039
    Figure imgb0040
  • Each forward primer contains a 17 base sequence to restore the beta-lactamase promoter on pBR322, and each reverse primer contains BsaI site (for NDM-1, OXA-48, SHV-18, KPC-3, IMP-4 and CTX-M15) or FokI site (for VIM-1) to create 5'-ACCG four base protruding 5' end. After amplifying each beta lactamase gene with high fidelity DNA polymerase such as Q5 DNA polymerase, the amplicon is digested with the appropriate restriction enzyme located in the reverse primer, described above. The digested amplicons are ready to ligate using T4 ligase between the Sspl and BsaI sites on the plasmid pBR322 (purchased from New England Biolabs), after removal of the TEM-3 fragment. SspI creates a blunt end and BsaI creates a 5'-CGGT protruding end. The reverse complement of the coding sequences of the each amplicons after restriction digestion are shown below. The 5' protruding end is underlined and 3' end of the promotor sequence is in bold small letters. The reverse complement CAT of the methionine initiating codons ATG of these seven genes, also shown in bold, yields a precise fusion of the coding region of the seven other beta-lactamases to the translational signal sequences of the TEM-3 beta-lactamase of pBR322.
    • NDM-1 (SEQ ID NO: 166)
      Figure imgb0041
    • OXA-48 (SEQ ID NO: 167)
      Figure imgb0042
    • SHV-18 (SEQ ID NO: 168)
      Figure imgb0043
    • VIM-1 (SEQ ID NO: 169)
      Figure imgb0044
    • KPC-3 (SEQ ID NO: 170)
      Figure imgb0045
    • IMP-4 (SEQ ID NO: 171)
      Figure imgb0046
    • CTX-M-15 (SEQ ID NO: 172)
      Figure imgb0047
      Figure imgb0048
  • Then DH5α competent cells purchased from New England Biolabs are transformed with these ligations followed by selection for the desired recombinants on LB Ampicillin (100µg/mL) plates.
  • Plasmid DNA samples are isolated from these transformants and submitted to DNA sequence analysis to confirm that the correct sequence for each of the seven different beta lactamases genes is present in each construct giving the plasmids:
    These pBR322 derivative plasmids so derived are named:
    1. i. pNB010 carrying the SHV-18 gene;
    2. ii. pNB011 carrying the CTX-M-15 gene;
    3. iii. pNB012 carrying the KPC-3 gene;
    4. iv. pNB013 carrying the VIM-1 gene;
    5. v. pNB014 carrying the IMP gene;
    6. vi. pNB015 carrying the NDM-1 gene;
    7. vii. pNB016 carrying the OXA-48 gene; in addition to, as described,
    8. viii. pBR322 carrying the TEM-3 gene
  • Then 7 recipient E. coli strains, DH5α, each carrying one of these seven beta lactamase genes on the plasmids pNB010-016 are subsequently transformed with the plasmids pNB104A, pNB104B, pNB108 and pNB203 and selected for chloramphenicol resistance to select for acquisition of these Nemesis symbiotic plasmids, along with the negative control pNB100 as well as with transformation of DH5α (pBR322) by pNB102 as the positive control, and then tested for conversion to ampicillin sensitivity as described in Example 2 (see Fig 14.)
    • "Nemesis symbiotic activity" (NSA) assay by plasmid conjugation to test activity of constructs carrying multiple spacers targeting beta lactamase gene families
  • The plasmid conjugation assay described in Example 2 may also be used to test the Nemesis symbiotic activity of the new CRISPR/Cas9 plasmid constructs carrying spacer sequences targeting multiple families of beta lactamase genes. The assay involves mating a donor cell carrying the beta lactamase gene on a conjugative plasmid, and hence ampicillin resistant, with a recipient cell carrying the Nemesis symbiotic on a non-mobilisable plasmid encoding chloramphenicol resistance. Exconjugants are selected on LB plates containing both 100µg/mL ampicillin and 35µg/mL chloramphenicol. Successful Nemesis symbiotic activity is seen in a reduction in the efficiency of transfer of the ampicillin resistance gene.
  • As donors, the same strain used in Example 2 was used. This strain, JA200 (F+ thr-1, leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl), also carries plasmid pNT3 as described by Saka et al. (). The plasmid pNT3 is a mobilisable plasmid carrying the TEM-1 beta lactamase gene of Tn1. Conjugation of pNT3 and hence transfer of ampicillin resistance is effected by the transfer functions of the co-resident F+ plasmid.
  • The other potential donors are the set of E. coli and K. pneumoniae strains carrying representative genes from these seven different families of beta-lactam antibiotics that were purchased from Culture Collections, Public Health England, as described above. These strains need to be chloramphenicol sensitive in order to allow selection for exconjugants and were tested for growth on LB chloramphenicol 35µg/mL plates. NCTC13368, a K. pneumoniae strain carrying the SHV-18 gene; NCTC13438 a K. pneumoniae strain carrying the KPC-3 gene; NCTC13476 an E. coli strain carrying an uncharacterised IMP gene; NCTC13443 a K. pneumoniae strain carrying the NDM-1 gene and NCTC13442 a K. pneumoniae strain carrying the OXA-48 gene were all found to be resistant to chloramphenicol, but NCTC13353, an E. coli strain carrying the CTX-M-15 gene, and NCTC13440, a K. pneumoniae strain carrying the VIM-1 gene, were found to be chloramphenicol sensitive and were taken forward to be tested in matings with the recipients.
  • As recipients, the strains used in Example 2 serve as controls for testing the new plasmid constructs. Thus the negative control is DH5α (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ with the plasmid pNB100 encoding the CRISPR/Cas9 cassette but no spacer sequence targeting any antibiotic resistance gene; and the positive control is DH5α with the plasmid pNB102 encoding the CRISPR/Cas9 cassette as well as the spacer sequence targeting the TEM beta-lactamase family. To be tested are DH5α strains with the plasmids pNB104A, pNB104B and pNB108. These plasmids also encode the CRISPR/Cas9 cassette in addition to the spacer sequences, all driven off one promotor in the following order, proximal to distal from the promotor (pNB104A): NDM-IMP-VIM-KPC; (pNB104B): OXA-48-SHV-TEM-CTX-M (pNB108): NDM-IMP-VIM-KPC-OXA-48-SHV-TEM-CTX-M.
  • To set up the matings, colonies of donors were picked from LB Ap100 (ampicillin 100µg/mL) and recipients from LB Cm35 (chloramphenicol 35µg/mL) plates into 200µL of LB and then mixed 100µL of recipients with 35µL of donors, then 5µL of the mixture were spotted onto LB plates and incubated at 37ºC for 5 hours to allow mating.
  • Figure 30A shows the results from mating the donor carrying the TEM-1 beta lactamase (JA200 (F+ thr-1, leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl)), and designated (5) in the figure, with the recipients DH5α pNB100, designated (-), DH5α pNB102, designated (+). Here a loopful of each mating mixture was resuspended in 220µL of LB and 200µL of the cells were plated on LB Ap100Cm35 plates to select for exconjugants. As expected from Example 2, the cross between the donor of TEM-1 and the recipient with pNB100 gives efficient mating and a lawn of cells (Fig. 30A, top left, 5-), and mating the donor of TEM-1 with the recipient with pNB102 encoding the single TEM spacer shows strong inhibition of transfer of the TEM-1 gene (see Fig. 30A, 5+, top right, where only a few isolated colonies are seen). The mating of the donor carrying the TEM-1 gene with the recipient carrying pNB104 (see bottom plate, 5B, of the figure) also shows strong inhibition of transfer of the TEM-1 gene and demonstrates that in the pNB104B construct, carrying the four spacers OXA-48-SHV-TEM-CTX-M 4, the TEM spacer is active.
  • Figure 30B shows the results from mating the strain NCTC13440, a K. pneumoniae strain carrying the VIM-1, designated (2), and the strain NCTC13353, an E. coli strain carrying the CTX-M-15 gene, designated (4). Again a loopful of each such mating mixture was resuspended in 220µL of LB and 200µL of the cells were plated on LB Ap100Cm35 plates to select for exconjugants. The results show that these strains are able to act as donors and transfer the VIM-1 and the CTX-M-15 genes respectively to the DH5α pNB100 negative control recipient lacking Nemesis symbiotic activity (see Fig. 30B, top left and top right respectively). However transfer of the VIM-1 and the CTX-M-15 genes was found to be strongly inhibited in matings with recipients carrying spacers targeted against these genes: pNB104A and pNB104B (see plates 2A and 4B, bottom left and right respectively).
  • In another experiment, an equivalent mating was done to test Nemesis symbiotic activity in recipients carrying all 8 spacer sequences in the plasmid pNB108. Here cells were picked directly from the mating mixture and streaked onto LBAp100Cm35 plates. Figure 30C plate, top left, again shows successful mating of donors 2, 4 and 5 with the recipient carrying pNB100 (see 2_,4_ and 5_ in Fig. 30C). In all cases, however, mating with the recipient carrying pNB108 (see Fig. 30C, plate top right, 2/8, 4/8 and 5/8) gives strong inhibition of transfer of ampicillin resistance gene. And for comparison to a positive control in this assay the bottom plate with 5+ (see Fig. 30C) shows strong inhibition of transfer of TEM-1 ampicillin resistance gene to the recipient carrying pNB102.
  • The experiments reported above provide the proof-of-concept that, in the model organism, Escherichia coli, DNA constructs carrying the Cas9 CRISPR region plus a spacer region with sequences directed against a target region of the beta-lactamase gene can inactivate ampicillin resistance when delivered by naked DNA transformation and bacteriophage infection as well as prevent transfer of ampicillin resistance by plasmid conjugation. It is apparent that Nemesis symbiotics of the invention can be applied to pathogenic bacteria and for other antibiotic resistance genes.
    • SEQUENCE LISTING
    • <110> Nemesis Bioscience Ltd
    • <120> Therapeutic
    • <130> P/71337.WO01/MAR
    • <150> GB1406674.0
      <151> 2014-04-14
    • <150> GB1413719.4
      <151> 2014-08-01
    • <150> GB1418508.6
      <151> 2014-10-17
    • <160> 172
    • <170> PatentIn version 3.5
    • <210> 1
      <211> 12
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR05 bla gene sequence
    • <400> 1
      gatacgggag gg   12
    • <210> 2
      <211> 12
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR05 bla gene reverse strand
    • <400> 2
      ccctcccgta tc   12
    • <210> 3
      <211> 10
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR05 short bacterial off-target sequence
    • <400> 3
      cgatacggga   10
    • <210> 4
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR05 short bacterial off-target sequence
    • <400> 4
      cgatacggga g   11
    • <210> 5
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR05 short bacterial off-target sequence
    • <400> 5
      gatacgggag g   11
    • <210> 6
      <211> 19
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 bla gene sequence
    • <400> 6
      tgctcatcat tggaaaacg   19
    • <210> 7
      <211> 19
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 bla gene sequence reverse strand
    • <400> 7
      cgttttccaa tgatgagca   19
    • <210> 8
      <211> 12
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence
    • <400> 8
      tcattggaaa ac   12
    • <210> 9
      <211> 10
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence
    • <400> 9
      tgctcatcat   10
    • <210> 10
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence - 3
    • <400> 10
      gctcatcatt g   11
    • <210> 11
      <211> 13
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence
    • <400> 11
      ctcatcattg gaa   13
    • <210> 12
      <211> 17
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence
    • <400> 12
      ctcagcattg caaaacg   17
    • <210> 13
      <211> 15
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence
    • <400> 13
      tcatcattga aaaac   15
    • <210> 14
      <211> 12
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence
    • <400> 14
      tcattggaaa ac   12
    • <210> 15
      <211> 19
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 bla gene sequence
    • <400> 15
      tcgccagtta atagtttgc   19
    • <210> 16
      <211> 19
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 bla sequence reverse strand
    • <400> 16
      agcggtcaat tatcaaacg   19
    • <210> 17
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR30 short bacterial off-target sequence - 1
    • <400> 17
      tcgccagtta a   11
    • <210> 18
      <211> 12
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 short bacterial off-target sequence
    • <400> 18
      tcgccagtta at   12
    • <210> 19
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 short bacterial off-target sequence
    • <400> 19
      cgccagttaa t   11
    • <210> 20
      <211> 12
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 short bacterial off-target sequence
    • <400> 20
      cgccagttaa ta   12
    • <210> 21
      <211> 15
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 short bacterial off-target sequence
    • <400> 21
      cgccagctaa tagtt   15
    • <210> 22
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 short bacterial off-target sequence
    • <400> 22
      ccagttaata g   11
    • <210> 23
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR70 short bacterial off-target sequence
    • <400> 23
      taatagtttg c   11
    • <210> 24
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 bla sequence
    • <400> 24
      ccgcgccaca tagcagaact   20
    • <210> 25
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 bla sequence reverse strand
    • <400> 25
      agttctgcta tgtggcgcgg   20
    • <210> 26
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 26
      acatagcaga a   11
    • <210> 27
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 27
      cgcgccacat a   11
    • <210> 28
      <211> 13
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 28
      cgcgccacat agc   13
    • <210> 29
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 29
      gcgccacata g   11
    • <210> 30
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 30
      gccacatagc a   11
    • <210> 31
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 31
      acatagcaga a   11
    • <210> 32
      <211> 11
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 32
      catagcagaa c   11
    • <210> 33
      <211> 16
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90 short bacterial off-target sequence
    • <400> 33
      gcgccatata gcagaa   16
    • <210> 34
      <211> 20
      <212> RNA
      <213> Artificial Sequence
    • <220>
      <223> Artificial RNA sequence
    • <220>
      <221> stem_loop
      <222> (1)..(10)
    • <400> 34
      uagauaacua cgauacggga   20
    • <210> 35
      <211> 20
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> Artificial RNA sequence
    • <220>
      <221> stem_loop
      <222> (1)..(11)
    • <400> 35
      acuuuaaaag ugcucaucau   20
    • <210> 36
      <211> 20
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> Artificial RNA sequence
    • <220>
      <221> stem_loop
      <222> (3)..(13)
    • <400> 36
      acguugcgca aacuauuaac   20
    • <210> 37
      <211> 20
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> Artificial RNA sequence
    • <220>
      <221> stem_loop
      <222> (9)..(17)
    • <400> 37
      acuuuuaaag uucugcuaug   20
    • <210> 38
      <211> 861
      <212> DNA
      <213> Unknown
    • <220>
      <223> Beta-lactamase gene from pBR322
    • <400> 38
      Figure imgb0049
      Figure imgb0050
    • <210> 39
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 39
      ccgcgtaggc atgatagaaa 20
    • <210> 40
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 40
      acgttaaaca ccgccattcc   20
    • <210> 41
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 41
      gcgctggaga aaagcagcgg   20
    • <210> 42
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 42
      aagctgattg cccatctggg   20
    • <210> 43
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 43
      acgctcaaca ccgcgatccc   20
    • <210> 44
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 44
      aactacttac tctagcttcc   20
    • <210> 45
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 45
      ttgttgctga aggagttggg   20
    • <210> 46
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 46
      agcgaaaaac accttgccga   20
    • <210> 47
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 47
      ctgggaaacg gcactgaatg   20
    • <210> 48
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 48
      tgggttgttg gagagaaaac   20
    • <210> 49
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 49
      aaacacagcg gcacttctcg   20
    • <210> 50
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 50
      aaaattgaag ttttttatcc   20
    • <210> 51
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 51
      tggcagccgc agtggaagcc   20
    • <210> 52
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 52
      tcacagctac ttgaaggttc   20
    • <210> 53
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 53
      atcaaaactg gcagccgcaa   20
    • <210> 54
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 54
      atcaaaactg gcagccgcaa   20
    • <210> 55
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 55
      cagtactcca accccagcat   20
    • <210> 56
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 56
      tcacctggcc gcaaatagtc   20
    • <210> 57
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 57
      acaacggatt aacagaagca   20
    • <210> 58
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 58
      agaacatcag cgcttggtca   20
    • <210> 59
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 59
      ataacggctt gacccagtca   20
    • <210> 60
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 60
      ggcaaccaga atatcagtgg   20
    • <210> 61
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 61
      ggatgccggt gacgaacagc   20
    • <210> 62
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 62
      gctacagtac agcgataacg   20
    • <210> 63
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 63
      gacgttgcgt cagcttacgc   20
    • <210> 64
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 64
      aactacttac tctagcttcc   20
    • <210> 65
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 65
      ttgttgctga aggagttggg   20
    • <210> 66
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 66
      agcgaaaaac accttgccga   20
    • <210> 67
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 67
      acctttaaag tgctgctgtg   20
    • <210> 68
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 68
      tgggttgttg gagagaaaac   20
    • <210> 69
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 69
      aaacacagcg gcacttctcg   20
    • <210> 70
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 70
      aaaattgaag ttttttatcc   20
    • <210> 71
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 71
      ggtttgatcg tcagggatgg   20
    • <210> 72
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 72
      gtggattaac gttccgaaag   20
    • <210> 73
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 73
      cagcgacagc aaagtggcat   20
    • <210> 74
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 74
      cttgccacct acagtgcggg   20
    • <210> 75
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 75
      cccccaaagg aatggagatc   20
    • <210> 76
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 76
      caccaagtct ttaagtggga   20
    • <210> 77
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 77
      ataacggctt gacccagtca   20
    • <210> 78
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 78
      ctgggaaacg gaactgaatg   20
    • <210> 79
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 79
      acgttaaaca ccgccattcc   20
    • <210> 80
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 80
      aactacttac tctagcttcc   20
    • <210> 81
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 81
      ttgttgctga aggagttggg   20
    • <210> 82
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 82
      aaacacagcg gcacttctcg   20
    • <210> 83
      <211> 34
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 83
      ggctagttaa aaataaaatt gaagtttttt atcc   34
    • <210> 84
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 84
      ggtttgatcg tcagggatgg   20
    • <210> 85
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Spacer sequence
    • <400> 85
      ataacggctt gacccagtca   20
    • <210> 86
      <211> 195
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 86
      Figure imgb0051
    • <210> 87
      <211> 81
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 87
      Figure imgb0052
    • <210> 88
      <211> 54
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 88
      gggctggcaa gccacgtttg gtgggtctcg aaacggtttg atcgtcaggg atgg   54
    • <210> 89
      <211> 90
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 89
      Figure imgb0053
    • <210> 90
      <211> 90
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 90
      Figure imgb0054
    • <210> 91
      <211> 76
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 91
      Figure imgb0055
    • <210> 92
      <211> 76
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 92
      Figure imgb0056
    • <210> 93
      <211> 76
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 93
      Figure imgb0057
    • <210> 94
      <211> 76
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 94
      Figure imgb0058
    • <210> 95
      <211> 111
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 95
      Figure imgb0059
    • <210> 96
      <211> 111
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 96
      Figure imgb0060
    • <210> 97
      <211> 54
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 97
      gggctggcaa gccacgtttg gtggctcttc aaacgtccat cccacttaaa gact   54
    • <210> 98
      <211> 111
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 98
      Figure imgb0061
    • <210> 99
      <211> 1368
      <212> PRT
      <213> Streptococcus pyogenes M1 GAS
    • <400> 99
      Figure imgb0062
      Figure imgb0063
      Figure imgb0064
      Figure imgb0065
      Figure imgb0066
      Figure imgb0067
    • <210> 100
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 100
      tagataacta cgatacggga 20
    • <210> 101
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 101
      gatcgttgtc agaagtaagt   20
    • <210> 102
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 102
      actttaaaag tgctcatcat   20
    • <210> 103
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 103
      tttactttca ccagcgtttc   20
    • <210> 104
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 104
      attaatagac tggatggagg   20
    • <210> 105
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 105
      acaattaata gactggatgg   20
    • <210> 106
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 106
      gcaacaatta atagactgga   20
    • <210> 107
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 107
      cccggcaaca attaatagac   20
    • <210> 108
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 108
      aactacttac tctagcttcc   20
    • <210> 109
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 109
      acgttgcgca aactattaac   20
    • <210> 110
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 110
      tgtaactcgc cttgatcgtt   20
    • <210> 111
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 111
      atgtaactcg ccttgatcgt   20
    • <210> 112
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 112
      aacttacttc tgacaacgat   20
    • <210> 113
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 113
      agtcacagaa aagcatctta   20
    • <210> 114
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> Anti-protospacer sequence
    • <400> 114
      acttttaaag ttctgctatg   20
    • <210> 115
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 115
      aaactagata actacgatac gggag   25
    • <210> 116
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 116
      aaaactcccg tatcgtagtt atcta   25
    • <210> 117
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 117
      aaacacttta aaagtgctca tcatg   25
    • <210> 118
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 118
      aaaacatgat gagcactttt aaagt   25
    • <210> 119
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 119
      aaacacgttg cgcaaactat taacg   25
    • <210> 120
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 120
      aaaacgttaa tagtttgcgc aacgt   25
    • <210> 121
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 121
      aaacactttt aaagttctgc tatgg   25
    • <210> 122
      <211> 25
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence
    • <400> 122
      aaaaccatag cagaacttta aaagt   25
    • <210> 123
      <211> 81
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence for dual targets
    • <400> 123
      Figure imgb0068
    • <210> 124
      <211> 81
      <212> DNA
      <213> Unknown
    • <220>
      <223> Adaptor sequence for dual targets
    • <400> 124
      Figure imgb0069
    • <210> 125
      <211> 9326
      <212> DNA
      <213> Artificial
    • <220>
      <223> Plasmid sequence
    • <400> 125
      Figure imgb0070
      Figure imgb0071
      Figure imgb0072
      Figure imgb0073
      Figure imgb0074
      Figure imgb0075
    • <210> 126
      <211> 4107
      <212> DNA
      <213> Artificial
    • <220>
      <223> Cas9 coding sequence
    • <400> 126
      Figure imgb0076
      Figure imgb0077
      Figure imgb0078
      Figure imgb0079
    • <210> 127
      <211> 69
      <212> DNA
      <213> Artificial
    • <220>
      <223> -10 promoter in pCas9
    • <400> 127
      Figure imgb0080
    • <210> 128
      <211> 80
      <212> DNA
      <213> Artificial
    • <220>
      <223> Leader sequence in pCas9
    • <400> 128
      Figure imgb0081
    • <210> 129
      <211> 194
      <212> DNA
      <213> Artificial
    • <220>
      <223> Spacer sequence
    • <400> 129
      Figure imgb0082
    • <210> 130
      <211> 86
      <212> RNA
      <213> Artificial
    • <220>
      <223> tracrRNA
    • <400> 130
      Figure imgb0083
      Figure imgb0084
    • <210> 131
      <211> 91
      <212> DNA
      <213> Artificial
    • <220>
      <223> pre-crRNA
    • <400> 131
      Figure imgb0085
    • <210> 132
      <211> 11
      <212> RNA
      <213> Artificial
    • <220>
      <223> Cleaved tracrRNA after first processing event
    • <400> 132
      ggaaccauuc a 11
    • <210> 133
      <211> 75
      <212> DNA
      <213> Artificial
    • <220>
      <223> Cleaved tracrRNA
    • <400> 133
      Figure imgb0086
    • <210> 134
      <211> 55
      <212> DNA
      <213> Artificial
    • <220>
      <223> cleaved pre-crRNA
    • <400> 134
      aaugguccca aacacuuuua aaguucugcu augguuuuag agcuaugctg uuuug   55
    • <210> 135
      <211> 42
      <212> DNA
      <213> Artificial
    • <220>
      <223> Cleaved pre-crRNA after 2nd processing event
    • <400> 135
      acuuuuaaag uucugcuaug guuuuagagc uaugctguuu ug   42
    • <210> 136
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> crRNA complementary sequence within target beta-lactamase DNA sequence
    • <400> 136
      acttttaaag ttctgctatg   20
    • <210> 137
      <211> 38
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90-containing target beta-lactamase DNA sequence anti-protospacer strand
    • <400> 137
      ccaagtcaac ttttaaagtt ctgctatgtg gcgcggta   38
    • <210> 138
      <211> 38
      <212> DNA
      <213> Unknown
    • <220>
      <223> CR90-containing target beta-lactamase DNA sequence protospacer strand
    • <400> 138
      taccgcgcca catagcagaa ctttaaaagt tgacttgg   38
    • <210> 139
      <211> 20
      <212> DNA
      <213> Unknown
    • <220>
      <223> crRNA complementary sequence within target beta-lactamase DNA sequence - reverse strand
    • <400> 139
      catagcagaa ctttaaaagt   20
    • <210> 140
      <211> 4758
      <212> DNA
      <213> Streptococcus pyogenes
    • <400> 140
      Figure imgb0087
      Figure imgb0088
      Figure imgb0089
      Figure imgb0090
    • <210> 141
      <211> 276
      <212> DNA
      <213> Streptococcus pyogenes
    • <400> 141
      Figure imgb0091
    • <210> 142
      <211> 10
      <212> DNA
      <213> Streptococcus pyogenes
    • <400> 142
      ggtctccatt   10
    • <210> 143
      <211> 10
      <212> DNA
      <213> Streptococcus pyogenes
    • <400> 143
      ggtcccaaaa   10
    • <210> 144
      <211> 452
      <212> DNA
      <213> Artificial
    • <220>
      <223> Fragment 3, amplified from genomic DNA of S. pyrogenes strain SF370
    • <400> 144
      Figure imgb0092
    • <210> 145
      <211> 9578
      <212> DNA
      <213> Artificial
    • <220>
      <223> Sequence of the final construct pNB100
    • <400> 145
      Figure imgb0093
      Figure imgb0094
      Figure imgb0095
      Figure imgb0096
      Figure imgb0097
      Figure imgb0098
    • <210> 146
      <211> 386
      <212> DNA
      <213> Artificial
    • <220>
      <223> Amplified sequence with primer NB018 and NB019 from pNB100 as template
    • <400> 146
      Figure imgb0099
      Figure imgb0100
    • <210> 147
      <211> 744
      <212> DNA
      <213> Artificial
    • <220>
      <223> Modified CRISPR array in pNB200
    • <400> 147
      Figure imgb0101
    • <210> 148
      <211> 23
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 148
      ccaactacct ccccttgctt aac   23
    • <210> 149
      <211> 18
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 149
      ggtgactgat ggccacgt   18
    • <210> 150
      <211> 23
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 150
      gggctggcaa gccacgtttg gtg   23
    • <210> 151
      <211> 23
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 151
      ccgggagctg catgtgtcag agg   23
    • <210> 152
      <211> 43
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 152
      attgaaaaag gaagagtatg gaattgccca atattatgca ccc   43
    • <210> 153
      <211> 38
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 153
      agtcccgcta ggtctcaacc gtcagcgcag cttgtcgg   38
    • <210> 154
      <211> 42
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 154
      attgaaaaag gaagagtatg cgtgtattag ccttatcggc tg   42
    • <210> 155
      <211> 55
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 155
      agtcccgcta ggtctcaacc gctagggaat aattttttcc tgtttgagca cttct   55
    • <210> 156
      <211> 47
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 156
      attgaaaaag gaagagtatg cgttattttc gcctgtgtat tatctcc   47
    • <210> 157
      <211> 41
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 157
      agtcccgcta ggtctcaacc gttagcgttg ccagtgctcg a   41
    • <210> 158
      <211> 57
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 158
      attgaaaaag gaagagtatg ttaaaagtta ttagtagttt attggtctac atgaccg   57
    • <210> 159
      <211> 48
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 159
      agtcccgcta ggatgacctg gctgaccgct actcggcgac tgagcgat   48
    • <210> 160
      <211> 43
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 160
      attgaaaaag gaagagtatg tcactgtatc gccgtctagt tct   34
    • <210> 161
      <211> 39
      <212> DNA
      <213> Artificial
    • <220>
      <223> KPC-3 reverse primer
    • <400> 161
      agtcccgcta ggtctcaacc gttactgccc gttgacgcc   39
    • <210> 162
      <211> 60
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 162
      attgaaaaag gaagagtatg agcaagttat ctgtattctt tatatttttg ttttgtagca   60
    • <210> 163
      <211> 61
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 163
      agtcccgcta ggtctcaacc gttagttgct tagttttgat ggttttttac tttcgtttaa   60
    • c 61
    • <210> 164
      <211> 44
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 164
      attgaaaaag gaagagtatg gttaaaaaat cactgcgcca gttc   44
    • <210> 165
      <211> 47
      <212> DNA
      <213> Artificial
    • <220>
      <223> Primer sequence
    • <400> 165
      agtcccgcta ggtctcaacc gttacaaacc gtcggtgacg attttag   47
    • <210> 166
      <211> 834
      <212> DNA
      <213> Unknown
    • <220>
      <223> NDM-1 sequence
    • <400> 166
      Figure imgb0102
    • <210> 167
      <211> 819
      <212> DNA
      <213> Unknown
    • <220>
      <223> OXA-48 sequence
    • <400> 167
      Figure imgb0103
      Figure imgb0104
    • <210> 168
      <211> 882
      <212> DNA
      <213> Unknown
    • <220>
      <223> SHV-1 sequence
    • <400> 168
      Figure imgb0105
    • <210> 169
      <211> 822
      <212> DNA
      <213> Unknown
    • <220>
      <223> VIM-1 sequence
    • <400> 169
      Figure imgb0106
    • <210> 170
      <211> 903
      <212> DNA
      <213> Unknown
    • <220>
      <223> KPC-3 sequence
    • <400> 170
      Figure imgb0107
      Figure imgb0108
    • <210> 171
      <211> 762
      <212> DNA
      <213> Unknown
    • <220>
      <223> IMP-4 sequence
    • <400> 171
      Figure imgb0109
    • <210> 172
      <211> 897
      <212> DNA
      <213> Unknown
    • <220>
      <223> CTX-M-15 sequence
    • <400> 172
      Figure imgb0110

Claims (15)

  1. A delivery vehicle for introducing a polynucleotide into an antibiotic-resistant microorganism, wherein the delivery vehicle is selected from the group consisting of a plasmid (such as a conjugative plasmid or other plasmid replicon), a nucleotide vector, linear double-stranded DNA, a non-virulent bacteriophage and a lysogenic bacteriophage, and comprises a recombinant polynucleotide for inactivation of DNA carrying at least one antibiotic resistance gene encoding an enzyme which confers antibiotic resistance to a microrganism, wherein the recombinant polynucleotide comprises a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing multiple RNA guide molecules, wherein each RNA guide molecule:
    (i) is transcribed from its own promoter sequence;
    (ii) mediates the binding of a CRISPR associated (Cas) DNA-binding polypeptide or its functional equivalent or its modified version thereof to the at least one antibiotic resistance gene(s); and
    (iii) has a spacer sequence sufficiently complementary to a target DNA sequence of the at least one antibiotic resistance gene for the at least one antibiotic resistance gene to be targeted and inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof, and wherein:
    either the Cas DNA-binding polypeptide or a functional equivalent or a modified version thereof is a modified Cas DNA-binding polypeptide which creates a deletion and reseals the target DNA sequence to inactivate the at least one antibiotic resistance gene to prevent double-strand break (DSB)-induced killing of the microorganism, such that the microorganism becomes sensitised to an antibiotic, or
    the Cas DNA-binding polypeptide or a functional equivalent or a modified version thereof creates a DSB in the target DNA sequence to inactivate the at least one antibiotic resistance gene, and the delivery vehicle further comprises a nucleotide sequence encoding a gene product which prevents direct killing of the microorganism due to the generation of the DSB in the at least one antibiotic resistance gene, such that the microorganism becomes sensitised to an antibiotic.
  2. The delivery vehicle according to claim 1, further comprising a nucleic acid sequence which encodes the Cas DNA-binding polypeptide or its functional equivalent or its modified version.
  3. The delivery vehicle according to either of claim 1 or claim 2, in which the Cas DNA-binding polypeptide is Cas9 or a functional equivalent or a modified version thereof.
  4. The delivery vehicle according to any of the preceding claims, comprising a further RNA guide molecule which targets a gene involved in pathogenicity or other aspects of microbial metabolism, for example a gene involved in bacterial metabolism for biofilm production.
  5. The delivery vehicle according to any of the preceding claims, in which the modified Cas DNA-binding polypeptide comprises a recombinase catalytic domain to reseal target DNA sequence.
  6. The delivery vehicle according to any of claims 1 to 4, wherein the gene product that prevents direct killing of the microorganism is a protein encoded by a replicon subject to degradation due to the DSB caused by the Cas DNA-binding polypeptide, or an antitoxin that neutralises the effect of a toxin or killer function carried by a replicon on which the target DNA sequence is located.
  7. The delivery vehicle according to any of the preceding claims, in which the CRISPR array nucleic acid sequence has or transcribes one or more RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of one or more beta-lactamase genes, for example one or more or all of the genes selected from the group consisting of: NDM, VIM, IMP, KPC, OXA, TEM, SHV, CTX, OKP, LEN, GES, MIR, ACT, ACC, CMY, LAT, and FOX.
  8. A composition comprising the delivery vehicle according to any of claims 1 to 7.
  9. The composition according to claim 8, in which the composition is a pharmaceutical composition, a non-pathogenic microorganism such as a commensal bacterium for example in a probiotic formulation, or a dietary supplement.
  10. The composition according to either of claims 8 or 9, formulated for topical, enteral or parenteral administration.
  11. The composition according to any of claims 8 to 10, for use as a medicament.
  12. The composition according to any of claims 8 to 11, for use in the treatment or prevention of an infection caused by an antibiotic-resistant microorganism comprising one or more antibiotic resistance genes targeted by the RNA guide molecules of the recombinant polynucleotide.
  13. A method of inactivating antibiotic resistance in an antibiotic-resistant microorganism, the method comprising introducing into the microorganism the delivery vehicle according to any of claims 1 to 7.
  14. A host cell comprising the recombinant polynucleotide as defined in any of claims 1 to 7.
  15. The host cell according to claim 14, in which the host cell is a commensal bacterium.
EP15718982.0A 2014-04-14 2015-04-14 Therapeutic Revoked EP3132034B1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PL15718982T PL3132034T3 (en) 2014-04-14 2015-04-14 Therapeutic
EP20190714.4A EP3766971A1 (en) 2014-04-14 2015-04-14 Therapeutic
SI201531414T SI3132034T1 (en) 2014-04-14 2015-04-14 Therapeutic

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB1406674.0A GB201406674D0 (en) 2014-04-14 2014-04-14 Therapeutic
GB201413719A GB201413719D0 (en) 2014-08-01 2014-08-01 Therapeutic
GB201418508A GB201418508D0 (en) 2014-10-17 2014-10-17 Therapeutic
PCT/GB2015/051132 WO2015159068A1 (en) 2014-04-14 2015-04-14 Therapeutic

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP20190714.4A Division EP3766971A1 (en) 2014-04-14 2015-04-14 Therapeutic

Publications (2)

Publication Number Publication Date
EP3132034A1 EP3132034A1 (en) 2017-02-22
EP3132034B1 true EP3132034B1 (en) 2020-08-19

Family

ID=53008804

Family Applications (2)

Application Number Title Priority Date Filing Date
EP20190714.4A Withdrawn EP3766971A1 (en) 2014-04-14 2015-04-14 Therapeutic
EP15718982.0A Revoked EP3132034B1 (en) 2014-04-14 2015-04-14 Therapeutic

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP20190714.4A Withdrawn EP3766971A1 (en) 2014-04-14 2015-04-14 Therapeutic

Country Status (13)

Country Link
US (2) US20170037414A1 (en)
EP (2) EP3766971A1 (en)
JP (2) JP6849435B2 (en)
CN (1) CN106536739B (en)
AU (2) AU2015248621B2 (en)
CA (1) CA2945573A1 (en)
DK (1) DK3132034T3 (en)
ES (1) ES2827279T3 (en)
HU (1) HUE051354T2 (en)
PL (1) PL3132034T3 (en)
PT (1) PT3132034T (en)
SI (1) SI3132034T1 (en)
WO (1) WO2015159068A1 (en)

Families Citing this family (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6261500B2 (en) 2011-07-22 2018-01-17 プレジデント アンド フェローズ オブ ハーバード カレッジ Evaluation and improvement of nuclease cleavage specificity
US20150044192A1 (en) 2013-08-09 2015-02-12 President And Fellows Of Harvard College Methods for identifying a target site of a cas9 nuclease
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
US9340800B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College Extended DNA-sensing GRNAS
US9737604B2 (en) 2013-09-06 2017-08-22 President And Fellows Of Harvard College Use of cationic lipids to deliver CAS9
DK3066201T3 (en) 2013-11-07 2018-06-06 Editas Medicine Inc CRISPR-RELATED PROCEDURES AND COMPOSITIONS WITH LEADING GRADES
US20150166985A1 (en) 2013-12-12 2015-06-18 President And Fellows Of Harvard College Methods for correcting von willebrand factor point mutations
US10787654B2 (en) 2014-01-24 2020-09-29 North Carolina State University Methods and compositions for sequence guiding Cas9 targeting
WO2015155686A2 (en) 2014-04-08 2015-10-15 North Carolina State University Methods and compositions for rna-directed repression of transcription using crispr-associated genes
WO2016022363A2 (en) 2014-07-30 2016-02-11 President And Fellows Of Harvard College Cas9 proteins including ligand-dependent inteins
WO2016033298A1 (en) 2014-08-28 2016-03-03 North Carolina State University Novel cas9 proteins and guiding features for dna targeting and genome editing
NZ738068A (en) 2015-05-06 2019-07-26 Snipr Tech Ltd Altering microbial populations & modifying microbiota
EA201792663A1 (en) 2015-05-29 2018-04-30 Норт Каролина Стейт Юниверсити METHODS OF SCREENING BACTERIA, ARCHEAN, ALGAE AND YEAST WITH THE USE OF CRISPR NUCLEIC ACIDS
JP7051438B2 (en) 2015-06-15 2022-04-11 ノース カロライナ ステート ユニバーシティ Methods and Compositions for Efficient Delivery of Nucleic Acid and RNA-Based Antibacterial Agents
US11286480B2 (en) 2015-09-28 2022-03-29 North Carolina State University Methods and compositions for sequence specific antimicrobials
US12043852B2 (en) 2015-10-23 2024-07-23 President And Fellows Of Harvard College Evolved Cas9 proteins for gene editing
CN105463003A (en) * 2015-12-11 2016-04-06 扬州大学 Recombinant vector for eliminating activity of kanamycin drug resistance gene and building method of recombinant vector
US11542466B2 (en) 2015-12-22 2023-01-03 North Carolina State University Methods and compositions for delivery of CRISPR based antimicrobials
EP3219799A1 (en) 2016-03-17 2017-09-20 IMBA-Institut für Molekulare Biotechnologie GmbH Conditional crispr sgrna expression
US20200385715A1 (en) * 2016-05-11 2020-12-10 The Regents Of The University Of Colorado, A Body Corporate Compositions and methods for altering bacteria fitness
GB201609811D0 (en) 2016-06-05 2016-07-20 Snipr Technologies Ltd Methods, cells, systems, arrays, RNA and kits
KR20230095129A (en) 2016-08-03 2023-06-28 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Adenosine nucleobase editors and uses thereof
CN109804066A (en) 2016-08-09 2019-05-24 哈佛大学的校长及成员们 Programmable CAS9- recombination enzyme fusion proteins and application thereof
WO2018039438A1 (en) 2016-08-24 2018-03-01 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
KR102622411B1 (en) 2016-10-14 2024-01-10 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 AAV delivery of nucleobase editor
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
TW201839136A (en) 2017-02-06 2018-11-01 瑞士商諾華公司 Composition and method for treating hemochromatosis
WO2018165504A1 (en) 2017-03-09 2018-09-13 President And Fellows Of Harvard College Suppression of pain by gene editing
WO2018165629A1 (en) 2017-03-10 2018-09-13 President And Fellows Of Harvard College Cytosine to guanine base editor
CA3057192A1 (en) 2017-03-23 2018-09-27 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable dna binding proteins
CN108866092B (en) 2017-05-11 2022-12-27 中国科学院遗传与发育生物学研究所 Production of herbicide-resistant genes and uses thereof
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
EP3658573A1 (en) 2017-07-28 2020-06-03 President and Fellows of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (pace)
WO2019139645A2 (en) 2017-08-30 2019-07-18 President And Fellows Of Harvard College High efficiency base editors comprising gam
WO2019050902A1 (en) * 2017-09-05 2019-03-14 Adaptive Phage Therapeutics, Inc. Methods to determine the sensitivity profile of a bacterial strain to a therapeutic composition
JP2021500036A (en) 2017-10-16 2021-01-07 ザ ブロード インスティテュート, インコーポレーテッドThe Broad Institute, Inc. Use of adenosine base editing factors
US10760075B2 (en) 2018-04-30 2020-09-01 Snipr Biome Aps Treating and preventing microbial infections
AU2019262200A1 (en) * 2018-05-04 2020-12-17 Locus Biosciences, Inc. Methods and compositions for killing a target bacterium
EP3798304B1 (en) * 2018-05-22 2024-12-11 Jichi Medical University Antibacterial phage, treatment composition, disinfectant, food, kit for identifying bacteria, method for producing treatment composition, method for eliminating bacteria, method for identifying bacteria, and method for treating animals
WO2019226953A1 (en) 2018-05-23 2019-11-28 The Broad Institute, Inc. Base editors and uses thereof
JP2021525105A (en) * 2018-05-25 2021-09-24 ローカス バイオサイエンシーズ，インク． Methods and compositions for killing target bacteria
KR102131869B1 (en) * 2018-09-12 2020-07-09 기초과학연구원 Composition For Inducing Apoptosis of Cells With Modified Genes
US12264313B2 (en) 2018-10-01 2025-04-01 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for genome modification and alteration of expression
US12264330B2 (en) 2018-10-01 2025-04-01 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for killing target cells
US12203123B2 (en) 2018-10-01 2025-01-21 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for screening for variant cells
EP3861120A4 (en) 2018-10-01 2023-08-16 North Carolina State University RECOMBINANT TYPE I CRISPR-CAS SYSTEM
US11851663B2 (en) * 2018-10-14 2023-12-26 Snipr Biome Aps Single-vector type I vectors
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof
MX2021011325A (en) 2019-03-19 2022-01-06 Broad Inst Inc METHODS AND COMPOSITIONS TO EDIT NUCLEOTIDE SEQUENCES.
CN111378660B (en) * 2020-02-29 2021-08-06 浙江大学 A kind of sgRNA targeting tetracycline resistance gene tetA and its knockout vector, vector construction method and application
KR20230019843A (en) 2020-05-08 2023-02-09 더 브로드 인스티튜트, 인코퍼레이티드 Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US20230181659A1 (en) * 2020-05-14 2023-06-15 Cz Biohub Sf, Llc Phage-mediated delivery of genes to gut microbiome
CN114540389B (en) * 2020-11-26 2024-05-14 深圳华大生命科学研究院 Method for preparing genetically engineered virus and application thereof
US20220290213A1 (en) * 2021-02-11 2022-09-15 Brigham Young University Composition, method, and system for a rapid, real-time pentaplex pcr assay for major beta-lactamase genes
GB202209518D0 (en) 2022-06-29 2022-08-10 Snipr Biome Aps Treating & preventing E coli infections
CN118421634A (en) * 2024-07-02 2024-08-02 广州明迅生物科技有限责任公司 SgRNA sequence for cutting gene, expression vector and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014124226A1 (en) 2013-02-07 2014-08-14 The Rockefeller University Sequence specific antimicrobials

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2398918T3 (en) * 2005-08-26 2013-03-22 Dupont Nutrition Biosciences Aps A method and arrangement to vertically support outstanding electrical resistance elements
WO2010075424A2 (en) * 2008-12-22 2010-07-01 The Regents Of University Of California Compositions and methods for downregulating prokaryotic genes
WO2012164565A1 (en) 2011-06-01 2012-12-06 Yeda Research And Development Co. Ltd. Compositions and methods for downregulating prokaryotic genes
CN105658796B (en) * 2012-12-12 2021-10-26 布罗德研究所有限公司 CRISPR-CAS component systems, methods, and compositions for sequence manipulation
EP4074330A1 (en) * 2013-09-05 2022-10-19 Massachusetts Institute of Technology Tuning microbial populations with programmable nucleases
US9388430B2 (en) * 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
DK3066201T3 (en) * 2013-11-07 2018-06-06 Editas Medicine Inc CRISPR-RELATED PROCEDURES AND COMPOSITIONS WITH LEADING GRADES

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014124226A1 (en) 2013-02-07 2014-08-14 The Rockefeller University Sequence specific antimicrobials

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
AMI M. KABADI, DAVID G. OUSTEROUT, ISAAC B. HILTON AND CHARLES A. GERSBACH: "Multiplex CRISPR/Cas9-based genome engineeringfrom a single lentiviral vector", NUCLEIC ACIDS RES., vol. 42, no. 19, 13 August 2014 (2014-08-13), pages e147, XP055177310
ANONYMOUS: "ATCC Product Sheet Escherichia coli (ATCC® 47055™)", AMERICAN TYPE CULTURE COLLECTION, pages 1 - 2
ANONYMOUS: "Safety Data Sheet LB BROTH 10855001", THERMO FISHER SCIENTIFIC, 13 April 2020 (2020-04-13), pages 1 - 9, XP055825923
BIKARD DAVID, EULER CHAD, JIANG WENYAN, NUSSENZWEIG PHILIP M, GOLDBERG GREGORY W, DUPORTET XAVIER, FISCHETTI VINCENT A, MARRAFFINI: "Development of sequence-specific antimicrobials based onprogrammable CRISPR-Cas nucleases /HHS Author Manuscript/", NAT. BIOTECHNOL., vol. 32, 5 October 2014 (2014-10-05), pages 1146 - 1150, XP055826004
BRIAN CHAIKIND, JEFFREY L. BESSEN, DAVID B. THOMPSON, JOHNNY H. HU AND DAVID R. LIU: "A programmable Cas9-serine recombinase fusionprotein that operates on DNA sequences in mammalian cells", NUCLEIC ACIDS RESEARCH, vol. 44, no. 20, 2016, pages 9758 - 9770, XP055411362
CHRIS PROUDFOOT, ARLENE L. MCPHERSON, ANDREAS F. KOLB, W. MARSHALL STARK: "Zinc Finger Recombinases with Adaptable DNA Sequence Specificity", PLOS ONE, vol. 6, 29 April 2011 (2011-04-29), pages e19537, XP055826011
CITORIK ROBERT J, MIMEE MARK, LU TIMOTHY K: "Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases /NIH Author Manuscript/", NAT. BIOTECHNOL., vol. 32, 21 September 2014 (2014-09-21), pages 1 - 18, XP055825907
LAURA A. DEVER, TERENCE S, DERMODY: "Mechanisms of Bacterial Resistance to Antibiotics", ARCH INTERN MED, vol. 151, May 1991 (1991-05-01), pages 886 - 895, XP055826016
PETER KUHNERT, JACQUES NICOLET, AND JOACHIM FREY: "Rapid and Accurate Identification of Escherichia coli K-12 Strains", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 61, no. 11, November 1995 (1995-11-01), pages 4135 - 4139, XP055825940
ROBERT J CITORIK; MIMEE MARK; LU TIMOTHY K: "Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases Supplementary Material", NATURE BIOTECHNOLOGY, 21 September 2014 (2014-09-21), pages 1 - 14, XP055345843
SHENGDAR Q TSAI; NICOLAS WYVEKENS; CYD KHAYTER; JENNIFER A FODEN; VISHAL THAPAR; DEEPAK REYON; MATHEW J GOODWIN; MARTIN J ARYEE; K: "Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing /HHS Author Manuscript/", NAT. BIOTECHNOL., vol. 32, 25 April 2014 (2014-04-25), pages 569 - 576, XP055826007

Also Published As

Publication number Publication date
CN106536739A (en) 2017-03-22
CA2945573A1 (en) 2015-10-22
PT3132034T (en) 2020-11-12
HUE051354T2 (en) 2021-03-01
US20180362990A1 (en) 2018-12-20
AU2020277257A1 (en) 2021-01-07
SI3132034T1 (en) 2020-12-31
US20170037414A1 (en) 2017-02-09
JP2021013383A (en) 2021-02-12
WO2015159068A1 (en) 2015-10-22
JP6849435B2 (en) 2021-03-24
PL3132034T3 (en) 2021-04-06
DK3132034T3 (en) 2020-10-19
AU2015248621B2 (en) 2020-08-27
ES2827279T3 (en) 2021-05-20
EP3132034A1 (en) 2017-02-22
AU2015248621A1 (en) 2016-11-17
CN106536739B (en) 2021-08-03
EP3766971A1 (en) 2021-01-20
JP2017513489A (en) 2017-06-01

Similar Documents

Publication Publication Date Title
EP3132034B1 (en) Therapeutic
US20220016190A1 (en) Delivery vehicle
US12246061B2 (en) Sequence specific antimicrobials
Walthers et al. The response regulator SsrB activates expression of diverse Salmonella pathogenicity island 2 promoters and counters silencing by the nucleoid‐associated protein H‐NS
EP2847323B1 (en) A bacteriophage for biocontrol of salmonella and in the manufacturing or processing of foods
JP7141756B2 (en) Multi-host range bacteriophages with different tail fibers
JP2022058377A (en) Multiple host range bacteriophage with different tail fibers
JP2019010125A (en) Methods for reducing conjugative plasmids in bacteria
US20210032638A1 (en) Plasmid curing
US20210093679A1 (en) Engineered gut microbes and uses thereof

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20161031

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

TPAC Observations filed by third parties

Free format text: ORIGINAL CODE: EPIDOSNTIPA

INTG Intention to grant announced

Effective date: 20171027

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: NEMESIS BIOSCIENCE LTD.

GRAJ Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR1

GRAL Information related to payment of fee for publishing/printing deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR3

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1234437

Country of ref document: HK

17Q First examination report despatched

Effective date: 20180209

INTC Intention to grant announced (deleted)
TPAC Observations filed by third parties

Free format text: ORIGINAL CODE: EPIDOSNTIPA

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20200608

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602015057666

Country of ref document: DE

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1303973

Country of ref document: AT

Kind code of ref document: T

Effective date: 20200915

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20201013

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: ING. MARCO ZARDI C/O M. ZARDI AND CO. S.A., CH

REG Reference to a national code

Ref country code: FI

Ref legal event code: FGE

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Ref document number: 3132034

Country of ref document: PT

Date of ref document: 20201112

Kind code of ref document: T

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20201105

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: NO

Ref legal event code: T2

Effective date: 20200819

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201119

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201120

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201219

REG Reference to a national code

Ref country code: HU

Ref legal event code: AG4A

Ref document number: E051354

Country of ref document: HU

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

REG Reference to a national code

Ref country code: DE

Ref legal event code: R026

Ref document number: 602015057666

Country of ref document: DE

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2827279

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20210520

PLBI Opposition filed

Free format text: ORIGINAL CODE: 0009260

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

PLAX Notice of opposition and request to file observation + time limit sent

Free format text: ORIGINAL CODE: EPIDOSNOBS2

REG Reference to a national code

Ref country code: FI

Ref legal event code: MDE

Opponent name: ELIGO BIOSCIENCE

26 Opposition filed

Opponent name: ELIGO BIOSCIENCE

Effective date: 20210518

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

PLBB Reply of patent proprietor to notice(s) of opposition received

Free format text: ORIGINAL CODE: EPIDOSNOBS3

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210414

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IE

Payment date: 20220209

Year of fee payment: 8

Ref country code: GB

Payment date: 20220224

Year of fee payment: 8

Ref country code: CH

Payment date: 20220215

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201219

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20220210

Year of fee payment: 8

Ref country code: PL

Payment date: 20220211

Year of fee payment: 8

Ref country code: NL

Payment date: 20220215

Year of fee payment: 8

Ref country code: IT

Payment date: 20220310

Year of fee payment: 8

Ref country code: FR

Payment date: 20220209

Year of fee payment: 8

Ref country code: BE

Payment date: 20220321

Year of fee payment: 8

PLAB Opposition data, opponent's data or that of the opponent's representative modified

Free format text: ORIGINAL CODE: 0009299OPPO

R26 Opposition filed (corrected)

Opponent name: ELIGO BIOSCIENCE

Effective date: 20210518

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PT

Payment date: 20220405

Year of fee payment: 8

Ref country code: NO

Payment date: 20220411

Year of fee payment: 8

Ref country code: HU

Payment date: 20220306

Year of fee payment: 8

Ref country code: ES

Payment date: 20220504

Year of fee payment: 8

Ref country code: DK

Payment date: 20220412

Year of fee payment: 8

Ref country code: DE

Payment date: 20220215

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20220413

Year of fee payment: 8

Ref country code: SI

Payment date: 20220228

Year of fee payment: 8

Ref country code: FI

Payment date: 20220412

Year of fee payment: 8

Ref country code: AT

Payment date: 20220325

Year of fee payment: 8

REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 1303973

Country of ref document: AT

Kind code of ref document: T

Effective date: 20200819

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602015057666

Country of ref document: DE

REG Reference to a national code

Ref country code: NO

Ref legal event code: MMEP

RDAF Communication despatched that patent is revoked

Free format text: ORIGINAL CODE: EPIDOSNREV1

REG Reference to a national code

Ref country code: DE

Ref legal event code: R103

Ref document number: 602015057666

Country of ref document: DE

Ref country code: DE

Ref legal event code: R064

Ref document number: 602015057666

Country of ref document: DE

Ref country code: DK

Ref legal event code: EBP

Effective date: 20230430

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20230501

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 1303973

Country of ref document: AT

Kind code of ref document: T

Effective date: 20230414

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20230414

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20230430

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230415

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230415

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231016

Ref country code: NO

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230501

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: HU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230415

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231103

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

REG Reference to a national code

Ref country code: SI

Ref legal event code: KO00

Effective date: 20240112

RDAG Patent revoked

Free format text: ORIGINAL CODE: 0009271

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: PATENT REVOKED

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

27W Patent revoked

Effective date: 20231127

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

REG Reference to a national code

Ref country code: AT

Ref legal event code: MA03

Ref document number: 1303973

Country of ref document: AT

Kind code of ref document: T

Effective date: 20231127

REG Reference to a national code

Ref country code: SI

Ref legal event code: NC00

Effective date: 20240704

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200819

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230414

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230415

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230415